In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto

ABSTRACT

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/291,054, filed Oct. 11, 2016, now U.S. Pat. No. 11,078,528, grantedon Aug. 3, 2021, which claims the benefit of U.S. ProvisionalApplication No. 62/240,347, filed Oct. 12, 2015, the entire contents ofwhich are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide sequence listing submitted concurrently herewith andidentified as follows: One 887 Byte ASCII (Text) file named“2021-06-29_38632-403_SQL_ST25” created on Jun. 29, 2021.

FIELD OF THE INVENTION

The present invention relates generally to detection of nucleic acids,and more specifically to in situ detection of nucleic acid variants.

BACKGROUND OF THE INVENTION

Recent studies revealed significant heterogeneity in tumor cellspreviously regarded as clones of each other (Gerlinger et al., N. Engl.J. Med. 366:883-892, 2012), meaning individual cancer cells in a tumorsite or tumor biopsy are not homogenous. In particular, neighboringcancer cells often have single nucleotide variations (SNVs) in DNA orRNA. Precision medicine thus demands in situ detection of SNV in tissuebiopsies, in which the cellular structure and contents have to besubstantially preserved through the assay. The complex physiochemicalstructures in cells and overwhelming amount of non-target nucleic acidsand other molecules present a “high noise” environment, which can resultin high background and which requires a combination of high specificityand high sensitivity that has not been achieved by existing in situnucleic acid detection techniques.

SNV detection requires a single set of target probes (TPs) to capture asingle signal-generating complex (SGC). In the previously described“double-Z” probe design disclosed in U.S. Pat. Nos. 7,709,198 and8,658,361, however, multiple sets of TPs are hybridized to the target toprovide sufficient number of SGCs for generating a detectable signal.

Thus, there exists a need for methods to detect single nucleotidevariations or other nucleic acid variations at the single cell level insitu. The present invention satisfies this need, and provides relatedadvantages as well.

SUMMARY OF INVENTION

The invention relates to methods of in situ detection of a nucleic acidvariation of a target nucleic acid in a sample, including singlenucleotide variations or splice variants, and the like. The method cancomprise the steps of contacting the sample with a probe that detectsthe nucleic acid variation and a neighbor probe; contacting the samplewith pre-amplifiers that bind to the nucleic acid variation probe andneighbor probe, respectively; contacting the sample with a collaborationamplifier that binds to the pre-amplifiers; and contacting the samplewith a label probe system, wherein hybridization of the components formsa signal generating complex (SGC) comprising a target nucleic acid withthe nucleic acid variation, the probes and amplifiers; and detecting insitu signal from the SGC on the sample. The invention also providessamples, tissue slides, and kits relating to detection of nucleic acidvariations of a target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary configuration of a signal-generating complex(SGC) for in situ detection of a single nucleotide variation (SNV) in atarget nucleic acid. The SGC comprises single nucleotide variationprobe—SP; neighbor probe—NP; SP pre-amplifier—SPM, which containsmultiple SP collaboration anchors—SPCA; NP pre-amplifier—NPM, whichcontains multiple NP collaboration anchors—NPCA; collaborationamplifier—COM; label probe system—LPS, which can comprise a plurality oflabel amplifiers—LM and each of which in turn can bind to a plurality oflabel probes—LP.

FIGS. 2A and 2B exemplify detail and orientations of SP and NP. FIGS. 2Aand 2B show in detail the configuration, position and orientation of SPand NP. FIG. 2A shows that SP comprises a target anchor segment (SPAT)complementary to the target sequence containing the SNV, a pre-amplifieranchor segment (SPAP) complementary to a segment on the SP pre-amplifier(SPM); NP comprises a target anchor segment (NPAT) complementary to thetarget sequence adjacent to the segment on the target that contains theSNV, a pre-amplifier anchor segment (NPAP) complementary to a segment onthe NP pre-amplifier (NPM); and an optional spacer between SPAT and SPAPor between NPAT and NPAP. FIG. 2B illustrates examples of differentorientations of SP and NP with respect to the target and the entiresignal generating complex (SGC).

FIGS. 3A-3C show different orientations or positions of SPM and NPM.FIGS. 3A and 3B show two examples of different orientations or positionsof SPM and NPM with respect to the SPAP and NPAP. The configurationshown in FIG. 3C is the same as that shown in FIG. 3B, where FIG. 3Creflects the flexibility of nucleic acid molecules and the ability ofthe configuration in FIG. 3B to provide NPCAs and SPCAs near each otherfor binding to a COM.

FIGS. 4A, 4B and 4C show different orientations of COM binding sites incollaborative hybridization. FIGS. 4A, 4B and 4C show exemplaryconfigurations where a collaboration amplifier (COM) is hybridizedsimultaneously to an SP collaboration anchor (SPCA) on SPM and an NPcollaboration anchor (NPCA) on NPM. FIGS. 4A and 4B depict the twocorresponding segments on COM hybridized to SPCA and NPCA in the sameorientation. The configuration depicted in FIG. 4B can be regarded as aspecial case of that in FIG. 4A. In FIG. 4B, the SPCA and NPCA arepositioned with an offset. As a result, the spacer between the twocorresponding segments on COM can be shortened or even removed, whichcan enhance the collaborative hybridization effect. FIG. 4C depicts thetwo corresponding segments on COM hybridized to SPCA and NPCA in thereverse orientation.

FIGS. 5A and 5B show an exemplary configuration where twosignal-generating complexes (SGCs) are captured to a single nucleotidevariation (SNV). FIG. 5A depicts both SGCs formed and binding to atarget nucleic acid. FIG. 5B depicts the binding of one SGC, whereas thesecond SGC is not bound. The lack of binding of the second SGC can bedue to an issue with probe access or target nucleic acid degradation. Inthe depicted configuration, detectable signal is still generated withone SGC bound to the target nucleic acid.

FIGS. 6A and 6B show exemplary embodiments where the number of assaysteps are reduced. The assay steps can be reduced by pre-assemblingcomponents of the SGC. FIG. 6A shows an embodiment in which the SPM andCOM are integrated, and the NPM and COM are integrated. In the depictedembodiment, the SPM is integrated with a COM and the NPM is integratedwith a COM using a “branched” molecule. FIG. 6B shows an embodiment inwhich the SP is integrated with the SPM, the NP is integrated with theNPM, and the COM is integrated with the LM.

FIGS. 7A and 7B show exemplary embodiments where collaborativehybridization is moved to different layers within SGC. FIG. 7A shows anSGC formed with collaborative hybridization between the COM and LM. FIG.7B shows an SGC formed with collaborative hybridization between the LMand LP.

FIGS. 8A and 8B show an exemplary embodiment incorporating more than onecollaborative hybridization steps in the assembly of the SGC during theassay. FIG. 8A shows a first collaborative hybridization between SPM orNPM and the SP and TP, respectively. A second collaborativehybridization is shown between the SPM, NPM and COMs. FIG. 8B shows afirst collaborative hybridization between SP, NP and the target nucleicacid, and a second collaborative hybridization between SPM, NPM andCOMs.

FIGS. 9A-9C show exemplary embodiments of detecting a specific splicejunction in a target nucleic acid sequence. FIG. 9A shows an exemplaryembodiment, where the SPAT hybridizes across the splice junction, thatis, it hybridizes to both nucleic acid segments brought together at thesplice junction, and the NPAT hybridizes to one of the nucleic acidsegments. FIG. 9B shows an exemplary embodiment, where the SPAThybridizes to one of the nucleic acid segments brought together at thesplice junction, and the NPAT hybridizes to the other nucleic acidsegment at the splice junction. FIG. 9C shows a configuration similar tothat depicted in FIG. 9B with respect to hybridization of SPAT and NPATto respective nucleic acid segments brought together at the splicejunction, where there is also hybridization between complementarysections of the NPAT and SPAT.

FIGS. 10A-10C show exemplary embodiments of utilizing splice junctiondetection methods to detect RNA molecules while avoiding the detectionof corresponding DNA. FIG. 10A shows an exemplary embodiment of RNAspecific detection using an exon junction bridging target probe. FIG.10B shows an exemplary embodiment of RNA specific detection using atarget probe set (nucleic acid variation probe and neighbor probe) thatcollaboratively hybridize to each other. FIG. 10C shows exemplaryembodiments of RNA specific detection using a target probe set (nucleicacid variation probe and neighbor probe).

FIGS. 11A, 11B and 11C show exemplary embodiments of detection of shortsequence.

FIGS. 12A and 12B show exemplary embodiments of detection of multipletargets. FIG. 12A shows a “pooling” approach where each target hasunique SPAT and NPAT in the SP-NP pair but the other elements are thesame in the rest of the SGC. A common signal is detected when any one ofthe target nucleic acids is present. FIG. 12B shows a “multiplexing”approach where each target nucleic acid has a unique SGC, which providesuniquely identifiable signals for each target nucleic acid.

FIGS. 13A-13D show the detection of BRAF V660E in sections of formalinfixed and paraffin embedded (FFPE) pellet of melanoma cell lines.Melanoma cell lines negative (CHL-1, a and a′, FIGS. 13A and 13B) andpositive (SK-MEL-28, b and b′, FIGS. 13C and 13D) for the V600E pointmutation of BRAF were assayed. Cells were hybridized to a target probesystem (TPS) containing the wild type detection probe (WDP) (a and b,FIGS. 13A and 13C) and a TPS containing a BRAF V600E mutation detectionprobe (MDP) (a′ and b′, FIGS. 13B and 13D) separately.

FIGS. 14A and 14B show the effect of SPATs of various lengths. FIG. 14Ashows a high number of false positives with the use of a long SPAT. FIG.14B shows that the assay has low sensitivity if the SPAT is too short.

FIGS. 15A and 15B show the effect of including modified bases in a SPAT.FIG. 15A shows staining results with normal bases in SPAT. FIG. 15Bshows improved results with modified bases used in SPAT.

FIGS. 16A-16D show detection of BRAF V600E in FFPE colon cancer tissuesknown to be negative (a and a′, FIGS. 16A and 16B) and positive (b andb′, FIGS. 16C and 16D) for the V600E point mutation. While signals wereobserved in both samples with probe targeting wild type BRAF mRNA (a andb, FIGS. 16A and 16C), V600E mutation mRNA was detected only in themutation positive sample with probe designed specifically for V600Emutation (b′, FIG. 16D).

FIGS. 17A and 17B show the detection of very low abundant and/ordegraded RNA target. FIG. 17A shows low staining of HGF target RNA,which is known to be very low abundant and partially degraded in thesample using RNA detection methods as previously described in U.S. Pat.Nos. 7,709,198 and 8,658,361. FIG. 17B shows improved staining of thesame target using a configuration similar to that shown in FIG. 11A.

FIGS. 18A and 18B show enhanced sensitivity of an exemplary method ofthe invention in detecting very short sequences compared to methodsdisclosed in U.S. Pat. Nos. 7,709,198 and 8,658,361. In FIG. 18A, thedetection system of U.S. Pat. Nos. 7,709,198 and 8,658,361 was used witha single pair of target probes to detect 50 nt sequence on POLR2A mRNAin Hela cell pellet. In FIG. 18B, a single SGC with a configurationsimilar to that shown in FIG. 11A was used to detect the same target inthe same sample type.

FIG. 19 demonstrates in situ detection of specific splice junctions,which can be used to identify a specific splice variant. Cell line H596is known to be META14 positive, that is, exon 14 in the MET gene is“skipped”, resulting in exon 15 splicing with exon 13 in MET RNA. Cellline A549 is the wild-type having all exons 12-15 in MET RNA. Probestargeting splicing junctions E12/13, E13/14 and E14/15 were used todetect the presence of corresponding junctions in FFPE (formalin fixedand paraffin embedded) cell pellets of H596 and A549 cells. The stainingimages are shown in FIG. 19, which shows sensitive and specificdetection of targeted splice junctions of E13/15 in H596 cells andE14/15 in A549 cells, showing that the META14 splice variant wascorrectly identified.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods that provide for highsensitivity detection of nucleic acid variants in a cell. The methodsare useful for detecting nucleic acid variations that can have clinicalimplications for disease status, disease progression, response totreatment of a disease, and the like. For example, cancers, includingtumors, are not homogeneous but rather can contain various types ofcells and/or cells of the same type but having different expressionlevels of proteins and nucleic acids between the cells. In some cases,there exist nucleic acid variations between the cells. Such nucleic acidvariations can include, but are not limited to, single nucleotidevariations, insertions and/or deletions (indels), splice variations,gene rearrangements, and the like. The methods and compositions of theinvention, as disclosed herein, can be used to detect nucleic acidvariants at the single cell level. Thus, the methods provide a highlysensitive and specific assay system to detect nucleic acid variations inclinical specimens, providing more detailed visualizable and clinicallyrelevant information on the expression of nucleic acid variations at thesingle cell level.

As described below in more detail, a probe is designed to detect nucleicacid variants, such as single nucleotide variations, insertions and/ordeletions, splice sites, gene rearrangements, and the like, and such aprobe is referred to herein as an SP. In embodiments of the invention,the SP can be a single nucleotide variant (SNV) probe, which can detecta single nucleotide variation in a target nucleic acid, or moregenerally a probe which can detect a specific nucleic acid variant thatinvolves more than one nucleotide, that is, a multi-nucleotide variant.Such multi-nucleotide variants would include a micro-insertion,micro-deletion, or modification of more than one nucleotide usingmethods such as CRISPER. In particular, a splice site or junction in atarget nucleic acid can be regarded as a special type ofmulti-nucleotide variant because this junction is specifically relatedto the nucleotides on each side of the junction. It is understood thatthe description of an SP herein or depiction in a figure of an SP hereincan be applied to any type of SP, with the SP designed to detect the SNVor multi-nucleotide variants such as a splice site. Thus, a descriptionherein or a configuration in a figure depicting the detection of an SNVusing an SP can be applied similarly to detection of multi-nucleotidevariants including a splice site, or other variant, with the SP designedto detect a multi-nucleotide variant rather than an SNV, with theremainder of the depicted configuration being applicable to detection ofthe multi-nucleotide variant in a target nucleic acid. Similarly, adescription herein or a depiction in a figure of detection of a splicesite can be applied to detection of SNV or multi-nucleotide variant,with the difference being whether the SP is designed to detect an SNV ormulti-nucleotide variant such as a splice site in a target nucleic acid.Thus, the description herein of an SP is understood to apply to thedetection of nucleotide variants in a target nucleic acid, depending onthe nature of the target nucleic acid.

The present invention provides a highly sensitive and specific in situdetection of nucleic acid variations in a high noise environment, forexample, tumor biopsies. In one embodiment, the nucleic acid variationis a single nucleotide variation (SNV). An exemplary embodiment of theinvention is shown in FIG. 1 and described below in more detail.

(1) SNV Probe (SP). As shown in FIG. 1 and in detail in FIG. 2A, asingle nucleotide variation (SNV) probe (SP) comprises twonon-overlapping regions, a target anchor segment (SPAT) and apre-amplifier anchor segment (SPAP), optionally separated by a spacer orlinker sequence. An SPAT is complementary to a target nucleic acidsequence encompassing the SNV site and has sufficient discriminatingpower to distinguish a single base change in the SNV sequence. Itslength and other parameters are designed to hybridize to targeted SNVbut not to the wild type or non-targeted SNV sequences. An SPAT isgenerally between about 10 to 20 nucleotides in length, while an SPAP isgenerally between about 14 to 28 nucleotides in length. The SP probedesign can be readily extended to detect insertions and deletions(indels) of 1-10,000 bases.

(2) Neighbor Probe (NP). Also shown in FIG. 1 and in detail in FIG. 2A,a neighbor probe (NP) comprises two non-overlapping regions, a targetanchor segment (NPAT) and a pre-amplifier anchor segment (NPAP),optionally separated by a spacer or linker sequence. An NPAT iscomplementary to a region of the target nucleic that is adjacent to theSNV and is generally between about 12 to 40 nucleotides in length. AnNPAP is generally between about 14 to 28 nucleotides in length.

The NP can sit left or right (5′ or 3′) to the SP bound to the targetSNV. In another embodiment, the NP and SP can assume different 5′ and 3′orientations in relationship to each other and in relationship to thesignal generating complex (SGC), as illustrated in FIG. 2. For example,as shown in FIG. 2B, the NP can have the NPAT on the 3′ and the SP canhave the SPAT on the 3′ end (FIG. 2B, upper left), the NP can have theNPAT on the 5′ end and the SP can have the SPAT on the 5′ end (FIG. 2B,upper right), the NP can have the NPAT on the 3′ end and the SP can havethe SPAT on the 5′ end (FIG. 2B, lower left), or the NP can have theNPAT on the 5′ end and the SP can have the SPAT on the 3′ end (FIG. 2B,lower right). It is possible to further enhance the specificity orsensitivity of mutation detection by incorporating multiple NPs in theadjacent region of the SNV. In one embodiment, two NPs sitting left andright (5′ or 3′, that is, flanking) to the SP bound to the target SNVcan be used to capture one or multiple SGCs and generate a detectablesignal (see FIG. 5A, showing two NPs flanking the SP).

The NP can bind stably to its complementary regions of the targetnucleic acid under the hybridization conditions employed. On the otherhand, the SPAT of SP is generally short (10-20 nucleotides) in order toenhance its power to discriminate SNV against non-SNV sequence. In oneembodiment, SPAT is shorter than NPAT or the melting temperature of SPATis lower than NPAT. A short SP can still hybridize to the target nucleicacid containing the SNV at the presence of NP due to the collaborativehybridization effect, that is, the melting temperature of the targetnucleic acid hybridizing to SP and NP simultaneously is higher than themelting temperature of the target nucleic acid hybridizing to SP or NPalone. The collaborative hybridization effect can be enhanced by targetprobe set configurations depicted in FIGS. 5 and 8, where in FIG. 5A,the SP is flanked by two NPs on both sides. In FIG. 8B, both SP and NPhave a third non-overlapping segment, that are complementary to eachother. In these cases, the melting temperature of the target nucleicacid hybridizing to SP and NP simultaneously is substantially higherthan the melting temperature of the target nucleic acid hybridizing toSP or NP alone. A detectable signal is generated when SP and NP arehybridized to adjacent regions of a single target nucleic acid, whereasthere is only weak or undetectable signal when SP and NP are nothybridizing to adjacent regions of a single target nucleic acid.

(3) Pre-amplifier for SP (SPM). As shown in FIG. 1 and in detail in FIG.3, a pre-amplifier for SP (SPM) comprises a single stranded nucleic acidof between about 50 and 500 nucleotides in length. The SPM comprises aplurality of repeat sequences between 10 and 20 nucleotides in lengthcalled an SP collaboration anchor (SPCA). SPM is linked to SP byhybridization. SPM comprises a segment complementary to thepre-amplifier anchor segment of SP (SPAP), which is designed to bind toSPAP in different positions or orientations as shown in FIG. 3. In apreferred embodiment, SPCA is repeated between 2 and 20 times in theSPM.

(4) Pre-amplifier for NP (NPM). As shown in FIG. 1 and in detail in FIG.3, a pre-amplifier for NP (NPM) comprises a single stranded nucleic acidof between about 50 and 500 nucleotides in length. The NPM comprises aplurality of repeat sequence between 10 and 20 nucleotides in lengthcalled an NP collaboration anchor (NPCA). NPM is linked to NP byhybridization. NPM comprises a segment complementary to thepre-amplifier anchor segment of NP (NPAP), which is designed to bind toNPAP in different positions or orientations as shown in FIG. 3. In apreferred embodiment, NPCA is repeated between 2 and 20 times in theNPM.

(5) Collaboration amplifier (COM). A collaboration amplifier (COM)comprises a single stranded nucleic acid of between about 60 and 900nucleotides in length. As shown in FIG. 1 and in more detail in FIG. 4,the COM comprises three non-overlapping segments, a segmentcomplementary to the SP collaboration anchor (SPCA) of the SPpre-amplifier (SPM), a segment complementary to the NP collaborationanchor (NPCA) of the NP pre-amplifier (NPM), and a segment containingrepeated sections, each of which can hybridize to a Label Probe System(LPS) that gives out signal for detection. The SPCA and NPCA hybridizeto COM in collaboration, that is, the melting temperature of COMhybridizing to SPCA and NPCA simultaneously is significantly higher thanthe melting temperature of COM hybridizing to SPCA or NPCA alone. Thatis, the hybridization condition employed in the assay is set as that aCOM cannot bind stably to either SPCA or NPCA alone. Since SPM and NPMbinds stably to SP and NP, respectively, COMs can stably bind to thetarget sequence when and only when both SP and NP are hybridized to thetarget nucleic acid and adjacent to each other. Since it is extremelyunlikely that such a unique configuration and positioning will occurnon-specifically, this collaboration hybridization significantly reducesfalse positive signal caused by non-specific binding of NPM or SPM in ahigh noise environment. The two segments complementary to SPCA and NPCAcan hybridize to them in different orientations, as depicted in FIG. 4.The segment of the COM that hybridizes to the LPS generally comprises aplurality of repeat sequences between about 15 and 30 nucleotides inlength, referred to as label amplifier anchor segments, that are capableof hybridizing to the LPS. In addition, the label amplifier anchorsegments can be located on either side or both sides of the segmentscomplementary to SPCA and NPCA.

(6) Label probe system (LPS). As shown in FIG. 1, a label probe system(LPS) hybridizes to COM. The label probe system (LPS) comprises aplurality of amplifiers, which are nucleic acids comprising a segmentthat can hybridize to complementary repeat sequences of the COM. Theamplifier also comprises a plurality of repeat sequences that canhybridize to a label probe (LP). The label probe comprises a nucleicacid comprising a segment that can hybridize to complementary repeatsequences of the amplifier. The label probe also comprises a detectablelabel. The label probe system thus provides a plurality of label probesbound to amplifiers, and a plurality of amplifiers can bind to a COM.

In more detail, an LPS comprises a plurality of label amplifiers (LMs)and a plurality of label probes (LPs), wherein each LM comprises asegment that can bind to a label amplifier anchor segment of the COM.The LM also comprises a plurality of label probe anchor segments. EachLP comprises a detectable label and a segment that hybridizes to thelabel probe anchor segment of the LM. When hybridizations occur betweenthe components, a signal generating complex (SGC) is formed. The SGCcomprises a target nucleic acid with the single nucleotide variation, anSP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, anda plurality of LPs (see FIG. 1).

The invention provides an assay of high specificity and sensitivity suchthat in situ detection of nucleic acid variations, including SNV,multi-nucleotide variants such as splice sites, and other variants, canbe performed in high noise samples. As illustrated in FIG. 1, an SGCprovides for very sensitive and specific detection of an SNV in a targetnucleic acid. As further illustrated in FIG. 1, a false signal can begenerated if a COM with LPS hybridized to it binds non-specifically to acomponent of the cell. However, the nature of the collaborativehybridization of the invention, as described herein, provides highsensitivity and specificity because the signal generated when bound tothe actual target is greater than the signal of COM boundnon-specifically. As described above, the SPCA is repeated at least 2times in the SPM, and the NPCA is repeated at least 2 times in the NPM.Such a configuration provides for a detectable signal of the actualtarget that is at least 2 times greater than that of a COM boundnon-specifically. The differential signal between COM-LPS bound to theactual target can be increased further, as described herein, byincreasing the number of SPCA repeats in the SPM and the number of NPCArepeats in the NPM (see also FIG. 1). In general, the number of SPCArepeats in the SPM will be the same as the number of NPCA repeats in theNPM, so that the COM can hybridize collaboratively to both the SPM andNPM. Therefore, the differential signal between binding of LPS to theactual target compared to non-specific binding can be increased toprovide a greater enhancement of signal to noise ratio, therebyproviding a higher specificity and higher sensitivity method to detectsingle nucleotide variations in individual cells in situ.

In one embodiment, the invention provides a method of in situ detectionof nucleic acid variations, for example, a single nucleotide variation,of a target nucleic acid. In an embodiment of the invention, provided isa method of in situ detection of a single nucleotide variation of atarget nucleic acid in a sample of fixed and permeabilized cells. Themethod can comprise the steps of: (A) contacting the sample with asingle nucleotide variation probe (SP) and a neighbor probe (NP),wherein the SP comprises a target anchor segment (SPAT) that canspecifically hybridize to a region of the target nucleic acid comprisingthe single nucleotide variation and a pre-amplifier anchor segment(SPAP), and wherein the NP comprises a target anchor segment (NPAT) thatcan hybridize to a region of the target nucleic acid adjacent to thebinding site of the SP and a pre-amplifier anchor segment (NPAP); (B)contacting the sample with an SP pre-amplifier (SPM) and an NPpre-amplifier (NPM), wherein the SPM comprises a segment that can bindto the SP and comprises two or more SP collaboration anchors (SPCAs),and wherein the NPM comprises a segment that can bind to the NP andcomprises two or more NP collaboration anchors (NPCAs); (C) contactingthe sample with a collaboration amplifier (COM), wherein the COMcomprises a first segment complementary to the SPCA, a second segmentcomplementary to the NPCA, and a third segment comprising a plurality oflabel amplifier anchor segments; (D) contacting the sample with a labelprobe system (LPS), wherein the LPS comprises a plurality of labelamplifiers (LMs) and a plurality of label probes (LPs), wherein each LMcomprises a segment that can bind to a label amplifier anchor segment ofthe COM and a plurality of label probe anchor segments, wherein each LPcomprises a detectable label and a segment that hybridizes to the labelprobe anchor segment of LM, wherein the aforesaid hybridizations form asignal generating complex (SGC) comprising a target nucleic acid withthe single nucleotide variation, an SP, an NP, an SPM, an NPM, aplurality of COMs, a plurality of LMs, and a plurality of LPs; and (E)detecting in situ signal from the SGC on the sample.

As described herein, the methods of the invention generally relate to insitu detection of nucleic acid variations. Methods for in situ detectionof nucleic acids are well known to those skilled in the art (see, forexample, US 2008/0038725; US 2009/0081688; Hicks et al., J. Mol. Histol.35:595-601 (2004)). As used herein, “in situ hybridization” or “ISH”refers to a type of hybridization that uses a directly or indirectlylabeled complementary DNA or RNA strand, such as a probe, to bind to andlocalize a specific nucleic acid, such as DNA or RNA, in a sample, inparticular a portion or section of tissue (in situ). The probe types canbe double stranded DNA (dsDNA), single stranded DNA (ssDNA), singlestranded complimentary RNA (sscRNA), messenger RNA (mRNA), micro RNA(miRNA), ribosomal RNA, mitochondrial RNA, and/or syntheticoligonucleotides. The term “fluorescent in situ hybridization” or “FISH”refers to a type of ISH utilizing a fluorescent label. The term“chromogenic in situ hybridization” or “CISH” refers to a type of ISHwith a chromogenic label. ISH, FISH and CISH methods are well known tothose skilled in the art (see, for example, Stoler, Clinics inLaboratory Medicine 10(1):215-236 (1990); In situ hybridization. Apractical approach, Wilkinson, ed., IRL Press, Oxford (1992);Schwarzacher and Heslop-Harrison, Practical in situ hybridization, BIOSScientific Publishers Ltd, Oxford (2000)).

For in situ detection of nucleic acid targets in a cell, the cell isoptionally fixed and permeabilized before hybridization of the targetprobes. Fixing and permeabilizing cells can facilitate retaining thenucleic acid targets in the cell and permit the target probes, labelprobes, amplifiers, preamplifiers, and so forth, to enter the cell. Thecell is optionally washed to remove materials not captured to a nucleicacid target. The cell can be washed after any of various steps, forexample, after hybridization of the target probes to the nucleic acidtargets to remove unbound target probes, after hybridization of thepreamplifiers, amplifiers, and/or label probes to the target probes,and/or the like. Methods for fixing and permeabilizing cells for in situdetection of nucleic acids, as well as methods for hybridizing, washingand detecting target nucleic acids, are also well known in the art (see,for example, US 2008/0038725; US 2009/0081688; Hicks et al., J. Mol.Histol. 35:595-601 (2004); Stoler, Clinics in Laboratory Medicine10(1):215-236 (1990); In situ hybridization. A practical approach,Wilkinson, ed., IRL Press, Oxford (1992); Schwarzacher andHeslop-Harrison, Practical in situ hybridization, BIOS ScientificPublishers Ltd, Oxford (2000)).

As used herein, the term “plurality” is understood to mean two or more.Thus, a plurality can refer to, for example, 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more,17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more,23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more,29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more,35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more,41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more,47 or more, 48 or more, 49 or more, 50 or more, 55 or more, 60 or more,65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more,95 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 ormore, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more,200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 ormore, 800 or more, 900 or more, or 1000 or more, or even a greaternumber, if desired for a particular use.

In one embodiment of the invention, the methods can be used to detect asingle nucleotide variation of a target nucleic acid. Such a singlenucleotide variation (SNV) can be a point mutation or asingle-nucleotide polymorphism (SNP). In an embodiment of a method ofthe invention, a sample containing cells is contacted with a singlenucleotide variation probe (SP) and a neighbor probe (NP). The SPcomprises a target anchor segment (SPAT) that can specifically hybridizeto a region of the target nucleic acid comprising the single nucleotidevariation. As used herein, an SP that can “specifically hybridize” to aregion of the target nucleic acid comprising the SNV refers to an SPthat can specifically hybridize to a target nucleic acid that containsthe SNV but not to a nucleic acid having a different nucleotide at theposition of the SNV. Thus, an SP can distinguish between a nucleic acidthat contains the SNV and a nucleic acid that does not contain the SNV.It is understood that an SP used in the methods and compositions of theinvention is designed such that, under the assay conditions utilized,the SP can specifically hybridize to a target nucleic acid containing aspecific nucleotide, such as the SNV, but will not hybridize to anucleic acid sequence containing a different nucleotide at thatposition, for example, a wild type nucleic acid sequence. Thus, an SP isselected to have an SPAT of a desired length suitable for exhibitingspecific hybridization to the target nucleic acid containing the SNVunder the temperature and buffers used for the in situ hybridizationassay. The length of the SPAT can be chosen to be sufficiently short inlength such that it will not remain stably bound to the target nucleicacid in the absence of binding of the NP. In general, the SPAT isrelatively short in length, for example, about 10 to 20 nucleotides inlength, but can be somewhat shorter or longer depending on the assayconditions used, such as about 9 to 21 nucleotides in length. Thus, ingeneral, an SPAT can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or21 nucleotides in length.

In the SP, the base complementary to the nucleotide variation on thetarget nucleic acid can be anywhere within the SPAT of the SP but isgenerally near the center of the SPAT. It is important that the SP beable to discriminate between the target nucleic acid having thenucleotide variation and a wild-type or other sequence that does notcontain the nucleotide variation. The SP should provide good sensitivityand specificity. To achieve this, the melting temperature differencebetween binding of the SP to a nucleic acid having the nucleotidevariation and to a wild-type or other sequence that does not contain thenucleotide variation (“dT_(m)”) should be maximized. The position of thebase within SPAT that is complementary to the nucleotide variation canbe selected to maximize dT_(m). This can be done by using meltingtemperature calculation algorithms known in the art (see, for example,SantaLucia, Proc. Natl. Acad. Sci. U.S.A. 95:1460-1465 (1998)). Inaddition, artificial modified bases such as Locked Nucleic Acid (LNA) orbridged nucleic acid (BNA) and naturally occurring 2′-O-methyl RNA areknown to enhance the binding strength between complementary pairs(Petersen and Wengel, Trends Biotechnol. 21:74-81 (2003); Majlessi etal., Nucl. Acids Res. 26:2224-2229 (1998)). These modified bases can bestrategically introduced into the SPAT of the SP to further increase thedT_(m) to enhance the detection sensitivity and specificity of SP.

One approach is to make all bases in the SPAT of the SP with modifiednucleotides (LNA, BNA or 2′-O-methyl RNA). Because each modified basecan increase the melting temperature, the length of SPAT can besubstantially shortened, which makes the SP more sensitive to a singlebase difference. Alternatively, only the base complementary to thenucleotide variation in the target nucleic acid is changed to themodified nucleotide. Because the binding strength of a modified base toits complement is stronger, the difference in melting temperatures(dT_(m)) is increased between the binding of SP to the nucleotidevariation in the target nucleic acid and the wild type or sequence thatdoes not contain the nucleotide variation. Yet another embodiment is touse three modified bases (for example, three LNA, BNA or 2′-O-methyl RNAbases, or a combination of two or three different modified bases) in theSPAT centered around the base complementary to the nucleotide variationin the target nucleic acid.

An SP also comprises a pre-amplifier anchor segment (SPAP). The SPAP iscomplementary to a segment of the SP pre-amplifier (SPM) and providesfor binding of the SPM to the SP bound to the target nucleic acid. TheSPAP is of a length that provides stable hybridization between the SPand SPM under the assay conditions used. Thus, the SPAP is generallylonger than the SPAT, for example, about 14 to 28 nucleotides in length,but can be somewhat shorter or longer depending on the assay conditionsused, such as about 10 to 30 nucleotides in length. Thus, in general, anSPAP can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 nucleotides in length.

The SP can optionally include a spacer between the SPAT and the SPAP.Thus, it is understood that an SP can have no spacer between the SPATand the SPAP. Generally, however, the SP will have a spacer between theSPAT and the SPAP. Such a configuration allows for a desired spatialseparation of the target nucleic acid from the SGC formed. A spacerbetween the SPAT and the SPAP will generally be 1 to 10 nucleotides inlength, but it is understood that the spacer can be longer, if desired.Thus, an optional spacer between the SPAT and the SPAP can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In aparticular embodiment, the spacer is 5 nucleotides in length.

In an embodiment of the invention, the sample is also contacted with aneighbor probe (NP). The NP comprises a target anchor segment (NPAT)that can hybridize to a region of the target nucleic acid adjacent tothe binding site of the SP. The region of the target nucleic acidadjacent to the SPAT binding site can be immediately adjacent, that is,it can bind with no gap between the SPAT binding site and the NPATbinding site. However, generally, there will be a gap of 1 to a fewnucleotides between the SPAT binding site and the NPAT binding site, forexample a gap of 1 to 50 nucleotides, and such binding sites will stillbe considered as adjacent binding sites for the SP and NP binding to thetarget nucleic acid.

The NP is selected to have an NPAT of a desired length suitable forexhibiting specific hybridization to the target nucleic acid adjacent tothe SPAT binding site under the temperature and buffers used for the insitu hybridization assay. Since the NP does not have to discriminatesingle nucleotide variation, the NPAT can be relatively longer than SPATto provide stability, for example, about 16 to 30 nucleotides in length,but can be somewhat shorter or longer depending on the assay conditionsused, such as about 12 to 40 nucleotides in length. Thus, in general, anNPAT can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides inlength.

An NP also comprises a pre-amplifier anchor segment (NPAP). The NPAP iscomplementary to a segment of the NP pre-amplifier (NPM) and providesfor binding of the NPM to the NP bound to the target nucleic acid. TheNPAP is of a length that provides stable hybridization between the NPand NPM under the assay conditions used. Thus, the NPAP is generallyabout 14 to 28 nucleotides in length, but can be somewhat shorter orlonger depending on the assay conditions used, such as about 10 to 30nucleotides in length. Thus, in general, an NPAP can be 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length.

The NP can optionally include a spacer between the NPAT and the NPAP.Thus, it is understood that an NP can have no spacer between the NPATand the NPAP. Generally, however, the NP will have a spacer between theNPAT and the NPAP. Such a configuration allows for a desired spatialseparation of the target nucleic acid from the SGC formed. A spacerbetween the NPAT and the NPAP will generally be 1 to 10 nucleotides inlength, but it is understood that the spacer can be longer, if desired.Thus, an optional spacer between the NPAT and the NPAP can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In aparticular embodiment, the spacer is 5 nucleotides in length.

In an embodiment of a method of the invention, the sample is contactedwith an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM). The SPMcomprises a segment that can bind to the SP by way of the SPAP. Thus,the segment of the SPM is complementary to the SPAP of the SP. Asdisclosed herein, the SPAP is generally of a length that provides stablehybridization between the SP and SPM under the assay conditions used.Thus, the segment of the SPM that is complementary to the SPAP isgenerally about 14 to 28 nucleotides in length, but can be somewhatshorter or longer depending on the length of the SPAP and the assayconditions used, such as about 10 to 30 nucleotides in length. Thus, ingeneral, the segment is complementary to the SPAP and can be 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length.

The SPM also comprises two or more SP collaboration anchors (SPCAs). TheSPCA provides a binding site for a collaboration amplifier (COM). Thelength of the SPCA is generally chosen to be sufficiently short inlength such that it will not remain stably bound to the COM in theabsence of binding of the COM to the NPCA of the NPM (see FIG. 1). Ingeneral, the SPCA is relatively short in length, for example, about 10to 20 nucleotides in length. Thus, in general, an SPCA can be 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. In aparticular embodiment, the SPCA is 14 nucleotides in length.

The NPM comprises a segment that can bind to the NP by way of the NPAP.

Thus, the segment of the NPM is complementary to the NPAP of the NP. Asdisclosed herein, the NPAP is generally of a length that provides stablehybridization between the NP and NPM under the assay conditions used.Thus, the segment of the NPM that is complementary to the NPAP isgenerally about 14 to 28 nucleotides in length, but can be somewhatshorter or longer depending on the length of the NPAP and the assayconditions used, such as about 10 to 30 nucleotides in length. Thus, ingeneral, the segment is complementary to the NPAP and can be 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length.

The NPM also comprises two or more NP collaboration anchors (NPCAs). TheNPCA provides a binding site for a collaboration amplifier (COM). Thelength of the NPCA is generally chosen to be sufficiently short inlength such that it will not remain stably bound to the COM in theabsence of binding of the COM to the SPCA of the SPM (see FIG. 1). Ingeneral, the NPCA is relatively short in length, for example, about 10to 20 nucleotides in length. Thus, in general, an NPCA can be 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. In aparticular embodiment, the NPCA is 14 nucleotides in length.

The SPM or NPM can optionally include a spacer between the SPCAs orNPCAs. Thus, it is understood that an SPM can have no spacer between theSPCAs, and an NPM can have no spacer between the NPCAs. Generally,however, the SPM and NPM will have a spacer between the SPCAs and NPCAs.An optional spacer between the SPCAs and NPCAs will generally be 1 to 10nucleotides in length, but it is understood that the spacer can belonger, if desired. Thus, an optional spacer between the SPCAs and NPCAscan be independently, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides in length. In a particular embodiment, the spacer is 5nucleotides in length. It is understood that the use of a spacer betweenany SPCAs of an SPM and NPCAs of an NPM is independent, and the lengthof the spacer between the SPCAs and NPCAs is independent. For example,if an NPM contains 4 NPCAs, there would be 3 optional spacers, and thosespacers do not have to be the same length as each other, that is, thelengths are independent.

As described herein, the SPM and NPM can be designed to contain two ormore SPCAs and NPCAs, respectively. To increase the signal associatedwith an SGC, the number of SPCAs and NPCAs can be increased (see FIG.1). The number of SPCAs and NPCAs can be selected to give a desiredsignal strength or increased signal to noise ratio. As shown in FIG. 1,each SPCA and NPCA can bind to a COM, which in turn binds to a labelprobe system (LPS). Increasing the number of SPCAs and NPCAs on the SPMand NPM, respectively, will result in a corresponding increase in thenumber of label probe systems (LPSs) bound to the target nucleic acid.Each COM that can bind to a nucleic acid target via collaborationhybridization in the methods of the invention increase thetarget-specific signal over non-specific binding of a COM to a componentof the cell (see FIG. 1). The number of SPCAs and NPCAs in a SPM and NPMpair will generally be the same between the pair, will generally havethe same spacing between the pairs, and will generally have 2 to 20SPCAs and NPCAs per pair, although it is understood that a higher numbercan be used, if desired. Thus, an SPM and NPM will generally have 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 SPCAs andNPCAs, respectively.

In an embodiment of a method of the invention, the sample is contactedwith a collaboration amplifier (COM). The COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments. As described herein and shown in FIG. 1, the COM providescollaboration hybridization between the SPM and NPM, which are bound tothe target nucleic acid via the SP and NP, respectively. Thecollaboration hybridization configuration of the COM provides additionalspecificity and a greater signal to noise ratio since the label probesystem (LPS) will not bind to the target nucleic acid unless the COM isbound to both the SPM and NPM. As discussed above, a further increase insignal is provided by increasing the number of SPCAs and NPCAs on theSPM and NPM, respectively. The collaborative hybridization of multipleCOMs is enhanced further by having multiple collaborative hybridizationreactions occur when the complex is specifically bound to the targetnucleic acid (see FIG. 1).

The COM also comprises a third segment, which comprises a plurality oflabel amplifier anchor segments. The label amplifier anchor segments arecomplementary to segments of the label amplifiers (LMs). The binding ofthe COM to the label amplifiers (LMs) is generally a stablehybridization. Thus, the label amplifier anchor segments are generallyof a length that provides stable hybridization between the COM and theLMs under the assay conditions used. The label amplifier anchor segmentsare generally about 20 to 28 nucleotides in length, but can be somewhatshorter or longer depending on the assay conditions used, such as about15 to 30 nucleotides in length. Thus, in general, the label amplifieranchor segments can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 nucleotides in length.

The length of a COM is selected based on the desired characteristics ofthe assay. As described above, the COM will contain a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments. The label amplifier anchor segments provide binding sites forlabel amplifiers (LMs). As shown in FIG. 4, the COM can optionallycontain a spacer independently between the first, second and/or thirdsegments. The length of the spacer and orientation of the first andsecond segment among SPCA and NPCA can be selected to maximize thecollaborative hybridization effect. The first and second segments of COMcan hybridize to SPCA and NPCA in the same or different orientations asshown in FIGS. 4A, 4B and 4C, respectively. The spacer is generally andindependently 1 to 10 nucleotides in length. Thus, a spacer between thefirst, second and/or third segments of a COM generally can be,independently, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length,depending on the configuration of the SGC.

The modified bases, such as LNA or BNA, can be used in SPCA, NPCA ortheir complementary sequences in COM, which increases the bindingstrength of the base to its complementary base, allowing an increase ofthe melting temperature of the hybridization between SPM and COM orbetween NPM and COM individually, or a reduction in the length of theanchoring segments (see, for example, Petersen and Wengel, TrendsBiotechnol. 21:74-81 (2003); U.S. Pat. No. 7,399,845). More importantly,such an approach substantially increases the difference between themelting temperatures of individual SPM-COM or NPM-COM hybridization andthe SPM-NPM-COM collaborative hybridization. Such a difference can beimportant to the enhancement of the signal to noise ratio in the assayof the invention because the binding of COM to an individual SPM or NPMis significantly more unstable than the binding of COM to a SPM/NPMpair. This ensures that an SGC can only be assembled when it isspecifically associated in the presence of the target.

Similarly, the third segment of a COM contains a plurality of labelamplifier anchor segments. The label amplifier anchor segments can alsooptionally and independently have a spacer between them. Thus, a spacerbetween the label amplifier anchor segments can be, independently, 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. Therefore, depending onthe length of the first, second and third segments, a COM will generallybe about 60 to 900 nucleotides in length.

In an embodiment of a method of the invention, the sample is contactedwith a label probe system (LPS). The LPS comprises a plurality of labelamplifiers (LMs). Each LM comprises a segment that can bind to a labelamplifier anchor segment of the COM. The LM also comprises a pluralityof label probe anchor segments. The binding of the LMs to the labelprobes (LPs) is generally a stable hybridization. Thus, the label probeanchor segments are generally of a length that provides stablehybridization between the LM and the LPs under the assay conditionsused. The label probe anchor segments are generally about 15 to 28nucleotides in length, but can be somewhat shorter or longer dependingon the assay conditions used, such as about 12 to 30 nucleotides inlength. Thus, in general, the label probe anchor segments can be 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length.

As described herein, to further increase the signal associated with anSGC, the number of LMs bound to a COM can be increased (see FIG. 1). Thenumber of LMs can be selected to give a desired signal strength orincreased signal to noise ratio. As shown in FIG. 1, a plurality of LMscan bind to a COM via the label amplifier anchor segments. Increasingthe number of label amplifier anchor segments on the COM will increasethe number of LMs bound to the COM. This in turn will result in acorresponding increase in the number of label probes bound to the targetnucleic acid. The number of LMs bound to the COM will generally be 2 to20, although it is understood that a higher number can be used, ifdesired. Thus, a COM will generally have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 label amplifier anchor segments,providing binding for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 LMs per COM.

The LPS also comprises a plurality of label probes (LPs). Each LPcomprises one or more detectable labels and a segment that hybridizes tothe label probe anchor segment of LM. As used herein, a “label” is amoiety that facilitates detection of a molecule. Common labels in thecontext of the present invention include fluorescent, luminescent,light-scattering, and/or colorimetric labels. Suitable labels includeenzymes, and fluorescent and chromogenic moieties, as well asradionuclides, substrates, cofactors, inhibitors, chemiluminescentmoieties, magnetic particles, rare earth metals, and the like. In aparticular embodiment of the invention, the label is an enzyme.Exemplary enzyme labels include, but are not limited to Horse RadishPeroxidase (HRP), Alkaline Phosphatase (AP), β-galactosidase, glucoseoxidase, and the like, as well as various proteases. Other labelsinclude, but are not limited to, fluorophores, Dinitrophenyl (DNP), andthe like. Labels are well known to those skilled in the art, asdescribed, for example, in Hermanson, Bioconjugate Techniques, AcademicPress, San Diego (1996), and U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labelsare commercially available and can be used in methods and assays of theinvention, including detectable enzyme/substrate combinations (Pierce,Rockford Ill.; Santa Cruz Biotechnology, Dallas Tex.; Invitrogen,Carlsbad Calif.). In a particular embodiment of the invention, theenzyme can utilize a chromogenic or fluorogenic substrate to produce adetectable signal, as described herein. Exemplary labels are describedherein.

Any of a number of enzymes or non-enzyme labels can be utilized so longas the enzymatic activity or non-enzyme label, respectively, can bedetected. The enzyme thereby produces a detectable signal, which can beutilized to detect a target nucleic acid. Particularly useful detectablesignals are chromogenic or fluorogenic signals. Accordingly,particularly useful enzymes for use as a label include those for which achromogenic or fluorogenic substrate is available. Such chromogenic orfluorgenic substrates can be converted by enzymatic reaction to areadily detectable chromogenic or fluorescent product, which can bereadily detected and/or quantified using microscopy or spectroscopy.Such enzymes are well known to those skilled in the art, including butnot limited to, horseradish peroxidase, alkaline phosphatase,β-galactosidase, glucose oxidase, and the like (see Hermanson,Bioconjugate Techniques, Academic Press, San Diego (1996)). Otherenzymes that have well known chromogenic or fluoregenic substratesinclude various peptidases, where chromogenic or fluorogenic peptidesubstrates can be utilized to detect proteolytic cleavage reactions. Theuse of chromogenic and fluorogenic substrates is also well known inbacterial diagnostics, including but not limited to the use of α- andβ-galactosidase, β-glucuronidase, 6-phospho-β-D-galatoside6-phosphogalactohydrolase, β-gluosidase, α-glucosidase, amylase,neuraminidase, esterases, lipases, and the like (Manafi et al.,Microbiol. Rev. 55:335-348 (1991)), and such enzymes with knownchromogenic or fluorogenic substrates can readily be adapted for use inmethods of the present invention.

Various chromogenic or fluorogenic substrates to produce detectablesignals are well known to those skilled in the art and are commerciallyavailable. Exemplary substrates that can be utilized to produce adetectable signal include, but are not limited to, 3,3′-diaminobenzidine(DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), Chloronaphthol(4-CN)(4-chloro-1-naphthol),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole(AEC) for horseradish peroxidase; 5-bromo-4-chloro-3-indolyl-1-phosphate(BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR/AS-MX), andp-Nitrophenyl Phosphate (PNPP) for alkaline phosphatase;1-Methyl-3-indolyl-θ-D-galactopyranoside and2-Methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside forβ-galactosidase; 2-Methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranosidefor β-glucosidase; and the like. Exemplary fluorogenic substratesinclude, but are not limited to, 4-(Trifluoromethyl)umbelliferylphosphate for alkaline phosphatase; 4-Methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-Methylumbelliferyl phosphate bis(cyclohexylammonium) and 4-Methylumbelliferyl phosphate forphosphatases; QuantaBlu™ and QuantaRed™ for horseradish peroxidase;4-Methylumbelliferyl β-D-galactopyranoside, Fluoresceindi(β-D-galactopyranoside) and Naphthofluoresceindi-(β-D-galactopyranoside) for β-galactosidase; 3-Acetylumbelliferylβ-D-glucopyranoside and 4-Methylumbelliferyl-β-D-glucopyranoside forβ-glucosidase; and 4-Methylumbelliferyl-α-D-galactopyranoside forα-galactosidase. Exemplary enzymes and substrates for producing adetectable signal are also described, for example, in US publication2012/0100540. Various detectable enzyme substrates, includingchromogenic or fluorogenic substrates, are well known and commerciallyavailable (Pierce, Rockford Ill.; Santa Cruz Biotechnology, Dallas Tex.;Invitrogen, Carlsbad Calif.; 42 Life Science; Biocare). Generally, thesubstrates are converted to products that form precipitates that aredeposited at the site of the target nucleic acid. Other exemplarysubstrates include, but are not limited to, HRP-Green (42 Life Science),Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green,Deep Space Black™, Warp Red™, Vulcan Fast Red and Ferangi Blue fromBiocare (Concord Calif.; biocare.net/products/detection/chromogens).

Biotin-avidin (or biotin-streptavidin) is a well known signalamplification system based on the fact that the two molecules haveextraordinarily high affinity to each other and that oneavidin/streptavidin molecule can bind four biotin molecules. Antibodiesare widely used for signal amplification in immunohistochemistry andISH. Tyramide signal amplification (TSA) is based on the deposition of alarge number of haptenized tyramide molecules by peroxidase activity.Tyramine is a phenolic compound. In the presence of small amounts ofhydrogen peroxide, immobilized Horse Radish Peroxidase (HRP) convertsthe labeled substrate into a short-lived, extremely reactiveintermediate. The activated substrate molecules then very rapidly reactwith and covalently bind to electron-rich moieties of proteins, such astyrosine, at or near the site of the peroxidase binding site. In thisway, a lot of extra hapten molecules conjugated to tyramide can beintroduced at the hybridization site in situ. Subsequently, thedeposited tyramide-hapten molecules can be visualized directly orindirectly. Such a detection system is described in more detail, forexample, in U.S. publication 2012/0100540.

Embodiments described herein can utilize enzymes to generate adetectable signal using appropriate chromogenic or fluorogenicsubstrates. It is understood that, alternatively, a label probe can havea detectable label directly coupled to the nucleic acid portion of thelabel probe. Exemplary detectable labels are well known to those skilledin the art, including but not limited to chromogenic or fluorescentlabels (see Hermanson, Bioconjugate Techniques, Academic Press, SanDiego (1996)). Exemplary fluorophores useful as labels include, but arenot limited to, rhodamine derivatives, for example,tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, TexasRed (sulforhodamine 101), rhodamine 110, and derivatives thereof such astetramethylrhodamine-5-(or 6), lissamine rhodamine B, and the like;7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and derivativesthereof; napthalenes such as dansyl(5-dimethylaminonapthalene-1-sulfonyl); coumarin derivatives such as7-amino-4-methylcoumarin-3-acetic acid (AMCA),7-diethylamino-3-[(4′-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA),Alexa fluor dyes (Molecular Probes), and the like;4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™) and derivativesthereof (Molecular Probes; Eugene Oreg.); pyrenes and sulfonated pyrenessuch as Cascade Blue™ and derivatives thereof, including8-methoxypyrene-1,3,6-trisulfonic acid, and the like; pyridyloxazolederivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow(3,6-disulfonate-4-amino-naphthalimide) and derivatives thereof; CyDye™fluorescent dyes (Amersham/GE Healthcare Life Sciences; PiscatawayN.J.), and the like. Exemplary chromophores include, but are not limitedto, phenolphthalein, malachite green, nitroaromatics such asnitrophenyl, diazo dyes, dabsyl (4-dimethylaminoazobenzene-4′-sulfonyl),and the like.

Well known methods such as microscopy, cytometry (mass cytometry,CyTOF), or spectroscopy can be utilized to visualize chromogenic orfluoroscent detectable signals associated with the respective targetnucleic acids. In general, either chromogenic substrates or fluorogenicsubstrates, or chromogenic or fluorescent labels, will be utilized for aparticular assay, if different labels are used in the same assay, sothat a single type of instrument can be used for detection of nucleicacid targets in the same sample.

As described herein, to further increase the signal associated with anSGC, the number of LPs bound to an LM can be increased (see FIG. 1). Thenumber of LPs can be selected to give a desired signal strength orincreased signal to noise ratio. As shown in FIG. 1, a plurality of LPscan bind to an LM via the label probe anchor segments. Increasing thenumber of label probe anchor segments on the LM will increase the numberof LPs bound to the LM. This results in an increase in the number oflabel probes bound to the target nucleic acid. The number of LPs boundto the LM will generally be 2 to 20, although it is understood that ahigher number can be used, if desired. Thus, an LM will generally have2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20label probe anchor segments, providing binding for 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 LPs per LM.

When the components described above hybridize, a signal generatingcomplex (SGC) is formed. The SGC comprises a target nucleic acid withthe nucleotide variation, for example, single nucleotide variation,multi-nucleotide variation, splice site, insertion/deletion,rearrangement, and the like, an SP, an NP, an SPM, an NPM, a pluralityof COMs, a plurality of LMs, and a plurality of LPs. Once the SGC isformed, the in situ signal can be detected from the SGC on the sample.After assembling each component, the sample can be optionally washed toremove unbound component prior to adding the next layer of component tothe sample.

The modified bases, such as LNA or BNA, can be used in the anchoringsegments of selected components of SGC, which increases the bindingstrength of the base to its complementary base, allowing a reduction inthe length of the anchoring segments (see, for example, Petersen andWengel, Trends Biotechnol. 21:74-81 (2003); U.S. Pat. No. 7,399,845).Artificial bases that expand the natural 4-letter alphabet such as theArtificially Expanded Genetic Information System (AEGIS; Yang et al.,Nucl. Acids Res. 34 (21): 6095-6101 (2006)) can be incorporated into thebinding sites among the interacting components of the SGC (for example,SPAP˜SPM, NPAP˜NPM, SPCA˜COM, NPCA˜COM, and LP˜LM hybridization sites).These artificial bases can increase the specificity of the interactingcomponents, which in turn can allow lower stringency hybridizationreactions to yield a higher signal.

It can be useful to use a configuration with an NP on each side of theSP, as shown in FIG. 5. Such a configuration provides for capture of twoSGCs to a target nucleic acid containing a nucleotide variant such as anSNV, a multi-nucleotide variant, splice site, insertion/deletion,rearrangement, and the like. Such a configuration can additionally beused to double the signal. Alternatively, it can also be used to enhancethe robustness of the assay. For example, as shown in FIG. 5B, if theaccess of one NP is blocked due to insufficient permeablization, or ifthe binding site to the target nucleic acid is lost due to RNAdegradation, a detectable signal can still be generated by the SGC boundto the other NP.

In an embodiment of the invention, the nucleic acid detected by themethods of the invention can be any nucleic acid present in the cellsample, including but not limited to, RNA, including messenger RNA(mRNA), micro RNA (miRNA), ribosomal RNA (rRNA), mitochondrial RNA, andthe like, or DNA, and the like. In a particular embodiment, the nucleicacid is RNA.

In a further embodiment of the invention, the fixed and permeabilizedcells are immobilized on a tissue slide. Methods for fixing andpermeabilizing cells for immobilizing on a tissue slide are well knownin the art, as disclosed herein.

As disclosed herein, the invention is based on building asignal-generating complex (SGC) bound to a target nucleic acid in orderto detect the presence of the target nucleic acid in the cell. Thecomponents for building an SGC generally comprise nucleic acids suchthat nucleic acid hybridization reactions are used to bind thecomponents of the SGC to the target nucleic acid. Methods of selectingappropriate regions and designing specific and selective reagents thatbind to the target nucleic acids, in particular oligonucleotides orprobes that specifically and selectively bind to a target nucleic acid,or other components of the SGC, are well known to those skilled in theart (see Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEd., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1999)). A desired specificity can be achieved using appropriateselection of regions of a target nucleic acid as well as appropriatelengths of a binding agent such as an oligonucleotide or probe, and suchselection methods are well known to those skilled in the art. Thus, oneskilled in the art will readily understand and can readily determineappropriate reagents, such as oligonucleotides or probes, that can beused to target one particular target nucleic acid over another targetnucleic acid, or to provide binding to the components of the SGC such asSP, NP, SPM, NPM, COM, LM and LP.

As disclosed herein, the steps of the methods of the invention, wherebycomponents are assembled into an SGC bound to a target nucleic acid, canbe performed concurrently or sequentially, in any order, so long as thetarget nucleic acid can be detected. In some cases, it can be desirableto reduce the number of assay steps, for example, reduce the number ofhybridization and wash steps. One way of reducing the number of assaysteps is to pre-assemble some or all components of the SGC prior tocontacting with a cell. Such a pre-assembly can be performed byhybridizing some or all of the components of the SGC together prior tocontacting the target nucleic acid. It is also possible to reduce theassay steps by pre-making some part of the SGC to integrate multiplecomponents of the SGC through chemical synthesis. One exemplaryembodiment is depicted in FIG. 6A. FIG. 6A shows the integration of SPMwith COM and NPM with COM using a pre-fabricated amplification moleculewith a “branched” structure. In this depicted embodiment, the SPM andNPM use a nucleic acid “branch” to connect to the COM, rather than anSPCA or NPCA as depicted in FIG. 6A. In this case, collaborative orcollaboration hybridization occurs at the binding of LM to the COM ofthe SPM-COM branched molecule.

Another embodiment is depicted in FIG. 6B. FIG. 6B shows integration ofSP and NP with the SMP and NPM, respectively, by extending the SP and NPto a longer sequence that includes SPCAs and NPCAs. The SGC can then beassembled with COMs, LMs and LPs, as depicted in FIG. 1, with the COMsbound to the SPCAs and NPCAs of the extended SP and NP, as depicted inFIG. 6B. Alternatively, the SGC can be assembled with the configurationdepicted in FIG. 6B, in which the COM and LM are integrated by using a“branched” configuration, as described above. The LM is connected to theCOM as a branched nucleic acid, rather than being bound by way of thelabel amplifier anchor segments, as depicted in FIG. 1. In this depictedembodiment, the collaborative hybridization occurs between the COMportion of the COM-LM branched molecule and the extended SP and NP,which contain SPCAs and NPCAs, respectively.

Large molecules are more prone to bind non-specifically or get stuck inthe cellular matrix during in situ assays. This is a potentialdisadvantage of using a “branched” molecule that provide large moleculesfor in situ detection assays. However, methods of the invention, asdisclosed herein, overcome this problem because a single largeamplification molecule cannot form the SGC alone. For example, in theembodiment shown in FIG. 6A, the SGC cannot form without the presence ofits pair (SP and NP bound to the target nucleic acid), so it will notgenerate noise or a false positive signal. In FIG. 6B, although a single“branched” large molecule can generate background noise ifnon-specifically bound, the intensity level of a true signal can beadjusted so that the number of labels bound to a target nucleic acid aregreater than that of a COM-LM branched molecule with bound label probes,as described herein (for example, increasing the number of SPCAs andNPCAs on the extended SP and TP).

Thus, it is understood that, if desired, an intermediary component canbe included such that the binding of one component to another ispre-assembled by chemical link. For example, in another embodiment, theLM can be a large pre-made molecule comprising many labels. In stillanother embodiment, the COM+LPS (COM/LM/LP) can be a chemicallysynthesized as a single large molecule. The use of such pre-made largemolecules can effectively reduce the number of assay steps. Methods ofmaking such nucleic acid configurations, including branched nucleic acidconfigurations as discussed above and shown in FIGS. 6A and 6B, are wellknown in the art (see, for example, U.S. Pat. Nos. 5,635,352 and5,681,697, which are incorporated herein by reference).

As disclosed herein, the components are generally bound directly to eachother. In the case of nucleic acid containing components, the bindingreaction is generally by hybridization. In the case of a hybridizationreaction, the binding between the components is direct. If desired, anintermediary component can be included such that the binding of onecomponent to another is indirect, for example, the intermediarycomponent contains complementary binding sites to bridge two othercomponents.

It is understood that the invention can be carried out in any desiredorder, so long as the variant target nucleic acid is detected. Thus, ina method of the invention, the steps of contacting a cell with an SP,NP, SPM, NPM, COM and/or LPS can be performed in any desired order, canbe carried out sequentially, or can be carried out simultaneously, orsome steps can be performed sequentially while others are performedsimultaneously, as desired, so long as the target nucleic acid isdetected. It is further understood that embodiments disclosed herein canbe independently combined with other embodiments disclosed herein, asdesired, in order to utilize various configurations, component sizes,assay conditions, assay sensitivity, and the like.

The methods of the invention, and related compositions, utilizecollaboration hybridization to increase specificity and to reducebackground in in situ detection of nucleic acid targets, where a complexphysiochemical environment and the presence of an overwhelming number ofnon-target molecules generates high noise. FIG. 1 illustrates anexemplary embodiment, where the collaboration hybridization is providedby the binding of a COM to an SPM and NPM. Using such a collaborationhybridization method, the binding of two or more COMs only occurs whenthe complex is bound to the target nucleic acid. As illustrated in FIG.1, this allows the method to be readily modified to provide a desiredsignal to noise ratio by increasing the number of COMs that can bind tothe target nucleic acid (that is, by increasing the number of SPCAs andNPCAs on the SPM and NPM, respectively).

In another embodiment, the collaboration hybridization can be applied toother components of the SGC as well. For example, the binding of LMs tothe COMs can be a stable reaction, as described herein, or the bindingcan be configured to require a collaboration hybridization. In such acase, the LM is designed such that the LM contains two segments thatbind to separate COMs. This configuration would be similar to therelationship of a COM to the SPM and NPM, but instead the LM would bindto a COM-1 and a COM-2. The LM and COMs are designed to have appropriatecomplementary segments, as with the COM, SPM and NPM depicted in FIG.7A, to provide collaboration hybridization.

Similarly, a further layer of collaboration hybridization can beutilized for binding of the LPs to the LM. In such a case, the LP isdesigned such that the LP contains two segments that bind to separateLMs. This configuration would be similar to the relationship of a COM tothe SPM and NPM, but instead the LP would bind to an LM-1 and an LM-2.The LP and LMs are designed to have appropriate complementary segments,as with the COM, SPM and NPM depicted in FIG. 7B, to providecollaboration hybridization.

Thus, the methods for detecting a target nucleic acid variation canutilize collaboration hybridization for the binding reactions betweenany one or all of the components in the detection system that providesan SGC specifically bound to a target nucleic acid. The number ofcomponents, and which components, to apply collaboration hybridizationcan be selected based on the desired assay conditions, the type ofsample being assayed, a desired assay sensitivity, and so forth. Any oneor combination of collaboration hybridization binding reactions can beused to increase the sensitivity and specificity of the assay.

FIG. 8 depicts two examples of SGC configurations that use collaborativehybridization between more than two components in the complex. In theconfiguration depicted in FIG. 8A, two collaborative hybridization stepshave to occur in order to build a stable SGC scaffold. In the depictedconfiguration, the SP is flanked by two NPs. A first collaborativehybridization occurs between the SPM/NPM and the SP and two NPs bound tothe target nucleic acid. A second collaborative hybridization occursbetween the COMs and SPM/NPM. Since collaborative hybridization enhancesspecificity and signal to noise ratio, as described herein, twocollaborative hybridization steps can further increase the assayrobustness in a high noise environment. FIG. 8B shows a differentapproach of utilizing two collaborative hybridization steps to improvespecificity. In this embodiment, the first collaborative hybridizationoccurs between the SP and NP, where the SP and NP are designed to havecomplementary sections. In this configuration, the SP and NP have threesegments, a first segment containing an SPAT or NPAT, which binds to thetarget nucleic acid (labeled “T” in FIG. 8B), a second segment thatcontains a complementary sequence to the respective NP or SP (labeled“P” in FIG. 8B), and a third segment containing an SPAP or NPAP, whichcan bind to the COM (labeled “L” in FIG. 8B). The SP and NP hybridize tothe target nucleic acid and to each other collaboratively in order forthe target probe set (SP and NP) and the target nucleic acid to form astable scaffold. Then SMP and NMP hybridize individually onto SP and NP,respectively, and the COMs hybridize collaboratively to the SP and NP.

As described herein, the configuration of various components can beselected to provide a desired stable or collaboration hybridizationbinding reaction. It is understood that, even if a binding reaction isexemplified herein as a stable or unstable reaction, any of the bindingreactions can be modified, as desired, so long as the target nucleicacid is detected. It is further understood that the configuration can bevaried and selected depending on the assay and hybridization conditionsto be used. In general, if a binding reaction is desired to be stable,the segments of complementary nucleic acid sequence between thecomponents is generally in the range of 16 to 30 nucleotides, orgreater. If a binding reaction is desired to be relatively unstable,such as when a collaboration hybridization binding reaction is employed,the segments of complementary nucleic acid sequence between thecomponents is generally in the range of 10 to 18 nucleotides. It isunderstood that the nucleotide lengths can be somewhat shorter or longerfor a stable or unstable hybridization, depending on the conditionsemployed in the assay. It is further understood, as disclosed herein,that modified nucleotides such as LNA or BNA can be used to increase thebinding strength at the modified base, thereby allowing length of thebinding segment to be reduced. Thus, it is understood that, with respectto the length of nucleic acid segments that are complementary to othernucleic acid segments, the lengths described herein can be reducedfurther, if desired. For example, microRNA is known to have shortsequence of about 22 nt. In order to use a SP-NP pair, a certain numberof modified nucleotides such as LNA or BNA can be incorporated in theSPAT and/or NPAT to reduce their length so that one or more SGCs can beassembled on a target microRNA.

The assay sensitivity can be further enhanced by selecting the number ofcomponents and binding reactions employed in the assay. As describedherein, in addition to providing one or more collaboration hybridizationbinding reactions, the signal can be increased by increasing the numberof SPCAs and NPCAs on the SPM and NPM, respectively. An increased numberof SPCAs and NPCAs provides for an increased number of COMs bound to thetarget nucleic acid. Similarly, the signal can also be increased byincreasing the number of label amplifier anchor segments on the COM. Anincreased number of label amplifier anchor segments provides for anincreased number of LMs bound to a COM. Further, the signal can beincreased by increasing the number of label probe anchor segments on theLM. An increased number of label probe anchor segments provide for anincreased number of LPs bound to an LM. It is understood that any ofthese options for increasing signal can be applied, as desired.Similarly and depending on application, the signal can be reduced byappropriately reducing the number of anchor segments mentioned above.Thus, the invention provides great flexibility for modifying theconfiguration of components of the assay to provide very sensitivedetection of a target nucleic acid. Alternatively, the signal level canalso be reduced by eliminating a layer of such intermediaryamplification molecules. For example, a plurality of LP can binddirectly to COM, eliminating LM. On the other hand, the signal level canalso be increased by adding one or more layers of such intermediaryamplification molecules.

As described herein, the present invention involves building a SGCaround a target nucleic acid containing a nucleotide variant such as anSNV, multi-nucleotide variant, splice site, or other nucleic acidvariation. The SGC comprises at least an SP that can specifically andsensitively bind to the nucleotide variant such as an SNV,multi-nucleotide variant, splice site, or other nucleic acid variation,of the target nucleic acid and that has sufficient discrimination suchthat the SP does not bind to non-target nucleic acids such as non-SNVsequences, non-multi-nucleotide variant sequences, non-splicedsequences, and the like. Once the SP binds to the SNV of the targetnucleic acid along with an NP, a large SGC is assembled with multiplelayers of amplification components, such as SPM/NPM, COM and LM on topof them, providing a large number of LPs to build up in the SGC togenerate sufficient signal to be detected. Such signal can present as adistinct “dot” in an imaging system. For example, if each SPM/NPM paircan carry A1 number of COM molecules, each COM can bind A2 number of LMmolecules, and each LM can bind A3 number of LP molecules, the totalnumber of LPs in an SGC is A1×A2×A3. Considering A1, A2 and A3 can eachbe as large as 20 or above, the total number of LPs in a SGC can be upto 8,000 and above. For the present invention, a “dot” in the stainingimage distinctively represents a target nucleic acid containing anucleotide variant such as an SNV, multi-nucleotide variant, splicesite, or other nucleotide variant, in the cell. The minimum number ofLPs needed in an SGC is determined by the sensitivity of the detectioninstrument as well as the amount of signal that can be generated by eachLP. In one embodiment where LP comprises fluorescent dyes, the minimumnumber of LPs in an SGC is at least 800, at least 1200, at least 1500,at least 2000, at least 2500 or at least 3000 to produce sufficientsignal to be detected by an instrument. In another embodiment, LPcomprises additional signal amplification, as described herein. Theminimum number of LPs needed in an SGC can be reduced to at least 300,at least 600, at least 1000 or at least 1500. Some components of the SGCscaffold may bind non-specifically or get stuck in a cell which may“collect” a certain number of LPs and generate a false positive signal.An important aspect of the invention is the collaborative hybridizationmechanisms built into the SGC structure so that any one component withinthe SGC scaffold that is non-specifically bound or stuck in a cellcannot generate detectable signal or at least only weak signal that canbe distinguished by the system as background noise. For example, if thecollaborative hybridization is built between COM and SPM+NPM, themaximum level of noise is generated when a COM molecule isnon-specifically bound or stuck with a false signal level at A2×A3 LPs.The theoretical signal to noise ratio of the assay is A1(=A1×A2×A3/A2×A3). If the collaborative hybridization is built betweenLM and COM, as shown in FIGS. 6A and 7A, the maximum false signal levelis generated when an LM is stuck with A3 number of LPs. The theoreticalsignal to noise ratio of the assay becomes A1×A2. Similarly, the assaysignal to noise ratio is A1×A2×A3 with the configuration shown in FIG.7B. In specific embodiments, this theoretical signal to noise ratio isat least 2, 4, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200.

As described previously, longer molecules with more repeats are neededto increase the signal generated by the SGC. However, larger moleculesmay have more difficulty penetrating into the cellular matrix to reachits hybridization sites and are harder to be washed out when they arestuck non-specifically in cell structures, thereby generating backgroundnoise. In addition, larger molecules are also more expensive to produce.It is therefore important to find optimized sizes for each layer ofamplification within. In one embodiment, A1 is relatively large because,as discussed previously, non-specific binding of SPM or NPM alone willnot produce false signal and larger A1 leads to a higher signal-to-noiseratio. In one embodiment of this invention, A1 is in the range of 4 to20. In another embodiment, A1 is preferably in the range of 6 to 16.Optionally, A2 is relatively smaller because non-specific binding ortrapping of a single COM can result in an assembly of A2×A3 LPsgenerating an equivalent level of background noise. In one embodiment,A2 is in the range of 3 to 15. In another embodiment, A2 is preferablyin the range of 5 to 12. Optionally, A3 can be relatively large comparedto A2, because non-specific binding or trapping of an LM molecule wouldproduce a relatively low level of noise proportional to A3. Anothermethod is to add one or more additional amplification layer in SGC inorder to reduce the length of molecule in individual layers.

In another embodiment, the invention provides a sample of fixed andpermeabilized cells, comprising (A) at least one fixed and permeabilizedcell containing a target nucleic acid with a single nucleotidevariation; (B) a single nucleotide variation probe (SP) comprising atarget anchor segment (SPAT) hybridized to a region of the targetnucleic acid comprising the single nucleotide variation, and, a neighborprobe (NP) comprising a target anchor segment (NPAT) hybridized to aregion of the target nucleic acid adjacent to the binding site of theSP; (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPMcomprises a plurality of SP collaboration anchors (SPCAs), and, an NPpre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises aplurality of NP collaboration anchors (NPCAs); (D) a plurality ofcollaboration amplifiers (COMs) each hybridized to the SPM and the NPM,wherein each COM comprises a first segment complementary to the SPCA, asecond segment complementary to the NPCA, and third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesingle nucleotide variation, an SP, an NP, an SPM, an NPM, a pluralityof COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGCprovides a signal that is detectable and distinguishable from thebackground noise.

In an additional embodiment, the invention provides a tissue slide,comprising (A) a slide having immobilized thereon a plurality of fixedand permeabilized cells comprising at least one fixed and permeabilizedcell containing a target nucleic acid with a single nucleotidevariation; (B) a single nucleotide variation probe (SP) comprising atarget anchor segment (SPAT) hybridized to a region of the targetnucleic acid comprising the single nucleotide variation, and, a neighborprobe (NP) comprising a target anchor segment (NPAT) hybridized to aregion of the target nucleic acid adjacent to the binding site of theSP; (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPMcomprises a plurality of SP collaboration anchors (SPCAs), and, an NPpre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises aplurality of NP collaboration anchors (NPCAs); (D) a plurality ofcollaboration amplifiers (COMs) each hybridized to the SPM and the NPM,wherein each COM comprises a first segment complementary to the SPCA, asecond segment complementary to the NPCA, and a third segment comprisinga plurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesingle nucleotide variation, an SP, an NP, an SPM, an NPM, a pluralityof COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGCprovides a signal that is detectable and distinguishable from thebackground noise.

In still another embodiment, the invention provides a kit for in situdetection of a single nucleotide variation of a target nucleic acid in asample of fixed and permeabilized cells, comprising (A) at least onereagent for permeabilizing cells; (B) a set of target hybridizing probescomprising a single nucleotide variation probe (SP) comprising a targetanchor segment (SPAT) capable of hybridizing to a region of the targetnucleic acid comprising the single nucleotide variation, and, a neighborprobe (NP) comprising a target anchor segment (NPAT) capable ofhybridizing to a region of the target nucleic acid adjacent to thebinding site of the SP; (C) a set of pre-amplifiers comprising an SPpre-amplifier (SPM) comprising a segment capable of hybridizing to theSP, wherein the SPM comprises a plurality of SP collaboration anchors(SPCAs), and, an NP pre-amplifier (NPM) comprising a segment capable ofhybridizing to the NP, wherein the NPM comprises a plurality of NPcollaboration anchors (NPCAs); (D) a collaboration amplifier (COM)capable of hybridizing to the SPM and the NPM, wherein the COM comprisesa first segment complementary to the SPCA, a second segmentcomplementary to the NPCA, and a third segment comprising a plurality oflabel amplifier anchor segments; (E) a label amplifier (LM) capable ofhybridizing to the label amplifier anchor segment of the COM, whereinthe LM comprises a plurality of label probe anchor segments; and (F) alabel probe (LP) capable of hybridizing to the label probe anchorsegment of the LM, wherein the LP comprises a detectable label; wherein,upon contacting a sample of fixed and permeabilized cells comprising acell containing a target nucleic acid with the single nucleotidevariation, the components in aforesaid (B)-(F) form a signal generatingcomplex (SGC) comprising the target nucleic acid with the singlenucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs,a plurality of LMs, and a plurality of LPs, and wherein the SGC providesa signal that is detectable and distinguishable from the backgroundnoise. The components of the kit can optionally be in a container, andoptionally instructions for using the kit can be provided.

As disclosed herein, the invention provides methods for detectingnucleic acid variations, such as single nucleotide variations (SNVs).Since the SP has the sensitivity and specificity to detect a singlenucleotide variation in a target sequence, it is understood that asimilar principle can be applied to detect other variations in targetnucleic acid or short sequences that involves more than one nucleotidesuch as RNA splicing variants, insertions, deletions, generearrangements and micro RNAs. Therefore, although exemplified asdetecting an SNV as a single nucleotide variation, it is understood thatthe methods can be applied to other types of nucleic acid variations.For example, the invention can be adopted to detect a unique junction,J, formed by two segments of nucleic acid sequences spliced together. Asdepicted in FIG. 9, a SP-NP probe set can be designed with SPAT spanningover the junction (FIG. 9A) or positioning the junction between NPAT andSPAT (FIG. 9B). FIG. 9C depicts another design similar to that in FIG.8B, in which SPAT is shortened and both NP and SP have a segment thatare complementary to each other. In this way, SP can only stablyhybridize to target when NP is present at the adjacent position.Alternatively, NPAT or both NPAT and SPAT can be shortened in thisdesign to achieve the same effect. In all three designs depicted in FIG.9, SP can only stably hybridize right next to NP to form SGC andgenerate detectable signal when and only when the two segments in thetarget splices together at the junction.

The capacity to detect the splicing of two nucleic acid segments hasmany applications. In the case of transcript or RNA splicing, a gene istranscribed into RNA, which is processed by RNA splicing to removeintrons and produce mRNA with contiguous exons providing a codingsequence. Some genes undergo alternative splicing, which can occurnaturally or in a disease state. In the case of an alternatively splicedgene, different exons are spliced together, resulting in a differentsequence. As with the detection of SNV, the methods of the invention canbe readily applied to detection of an alternatively spliced mRNA. Asdescribed herein, the methods are based on using a probe that isspecific to a target nucleic acid that detects the nucleic acidvariation. In the case of SNV, the variation is a single nucleotide,whereas in the case of an alternatively spliced gene, the sequencevariation can include numerous different nucleotides, that is, adifferent sequence, as a result of a different junction between twoexons. In this case, the SP probe can be designed to span the variantjunction sequence, preferably placing the junction near the center ofSPAT. Alternatively, the variant junction point can be placed betweenNPAT and SPAT.

In addition, to detecting splice variants, the methods of the inventioncan be used to detect gene rearrangements. It is well known that manycancers are characterized by gene rearrangements, in which oncogenes areactivated or growth repressors are inactivated. Similar totranscript/RNA splicing, the gene rearrangement results in a sequencevariation from a non-rearranged gene. When transcribed, the mRNA alsocontains the sequence variation as fusion transcripts. As with detectingRNA splicing, the methods of the invention can be readily applied todetect gene rearrangements or fusion transcripts. For example, the SPprobe can be designed to span the variant junction sequence in therearranged DNA or the fusion transcript, preferably placing the junctionpoint near the center of SPAT. Alternatively, the variant junction canbe placed between NPAT and SPAT. A similar strategy can be employed todetect insertions and deletions. For example, the point of an insertionor deletion can be placed within SPAT, preferably near the center ofSPAT. Alternatively, the point of insertion or deletion can be placedbetween NPAT and SPAT.

In a similar manner as detecting splice variants and gene rearrangments,the methods of the invention can be adopted to detect RNA specificallywithout detecting the corresponding DNA in the same cell. FIG. 10A showsa different approach, where the SPAT on SP bridges across the junctionof two adjacent exons. With the exons adjacent to each other in themRNA, SP can bind to the target nucleic acid right next to the NP,providing for assembly of the SGC on the target nucleic acid. With theexons spaced widely apart in DNA, the SP cannot bind to the targetnucleic acid, and therefore SGC cannot form, leading to the absence ofsignal.

Another embodiment is illustrated in FIG. 10B. In this embodiment, theSP and NP have collaborative hybridization with each other, as depictedin FIG. 8B, where the SP and NP can only stably bind to the targetnucleic acid when they hybridize to the target nucleic acid and eachother simultaneously. The SP and NP cannot bind to the DNA because theSP and NP binding sites are not adjacent as in the mRNA. As shown inFIG. 10B, the SP and NP are designed to each bind to separate exons atthe site of an exon junction. The SP and NP can only bind stably to anmRNA target because the exons to which the SP and NP bind are onlyadjacent in the mRNA. Therefore, a stable SGC can be formed on the mRNA,leading to detectable signal. In the case of the DNA, the SP and NPcannot stably bind to the target nucleic acid, because the respective SPand NP binding sites are not adjacent in the DNA. In this situation, noSGC can be formed (see FIG. 10B). Another embodiment is shown in FIG.10C, where the splice junction of exons are placed between SP and NP. Asshown in FIGS. 10A, 10B and 10C, exons are separated in DNA but splicedtogether in the mRNA transcript, forming unique junctions in mRNA, whichcan be readily detected using the methods depicted in FIG. 9. Since theexons are separated in DNA, no stable SP-NP set will be hybridizedadjacent to each other; no SGC can be formed, which leads to nodetectable signal, as shown in FIG. 10C.

In another embodiment, the invention provides a method of in situdetection of a spliced target nucleic acid in a sample of fixed andpermeabilized cells, comprising (A) contacting the sample with a splicesite probe (SP) and a neighbor probe (NP), wherein the SP comprises atarget anchor segment (SPAT) that can specifically hybridize to a regionof the target nucleic acid comprising the splice site and apre-amplifier anchor segment (SPAP), and wherein the NP comprises atarget anchor segment (NPAT) that can hybridize to a region of thetarget nucleic acid adjacent to the binding site of the SP and apre-amplifier anchor segment (NPAP); (B) contacting the sample with anSP pre-amplifier (SPM) and an NP pre-amplifier (NPM), wherein the SPMcomprises a segment that can bind to the SP and comprises two or more SPcollaboration anchors (SPCAs), and wherein the NPM comprises a segmentthat can bind to the NP and comprises two or more NP collaborationanchors (NPCAs); (C) contacting the sample with a collaborationamplifier (COM), wherein the COM comprises a first segment complementaryto the SPCA, a second segment complementary to the NPCA, and a thirdsegment comprising a plurality of label amplifier anchor segments; (D)contacting the sample with a label probe system (LPS), wherein the LPScomprises a plurality of label amplifiers (LMs) and a plurality of labelprobes (LPs), wherein each LM comprises a segment that can bind to alabel amplifier anchor segment of the COM and a plurality of label probeanchor segments, wherein each LP comprises a detectable label and asegment that hybridizes to the label probe anchor segment of LM, whereinthe aforesaid hybridizations form a signal generating complex (SGC)comprising a target nucleic acid with the splice site, an SP, an NP, anSPM, an NPM, a plurality of COMs, a plurality of LMs, and a plurality ofLPs; and (E) detecting in situ signal from the SGC on the sample. In oneembodiment, the SPAT can specifically hybridize to one of the twospliced nucleic acid segments. In another embodiment, the SPAT canspecifically hybridize to both of the two spliced nucleic acid segments.

In another embodiment, the invention provides a sample of fixed andpermeabilized cells, comprising (A) at least one fixed and permeabilizedcell containing a spliced target nucleic acid; (B) a splice site probe(SP) comprising a target anchor segment (SPAT) hybridized to a region ofthe target nucleic acid comprising the splice site, and, a neighborprobe (NP) comprising a target anchor segment (NPAT) hybridized to aregion of the target nucleic acid adjacent to the binding site of theSP; (C) an SP pre-amplifier (SPM) hybridized to the SP, wherein the SPMcomprises a plurality of SP collaboration anchors (SPCAs), and, an NPpre-amplifier (NPM) hybridized to the NP, wherein the NPM comprises aplurality of NP collaboration anchors (NPCAs); (D) a plurality ofcollaboration amplifiers (COMs) each hybridized to the SPM and the NPM,wherein each COM comprises a first segment complementary to the SPCA, asecond segment complementary to the NPCA, and third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesplice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, aplurality of LMs, and a plurality of LPs, and wherein the SGC provides asignal that is detectable and distinguishable from the background noise.

In another embodiment, the invention provides a tissue slide, comprising(A) a slide having immobilized thereon a plurality of fixed andpermeabilized cells comprising at least one fixed and permeabilized cellcontaining a spliced target nucleic acid; (B) a splice site probe (SP)comprising a target anchor segment (SPAT) hybridized to a region of thetarget nucleic acid comprising the splice site, and, a neighbor probe(NP) comprising a target anchor segment (NPAT) hybridized to a region ofthe target nucleic acid adjacent to the binding site of the SP; (C) anSP pre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises aplurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier(NPM) hybridized to the NP, wherein the NPM comprises a plurality of NPcollaboration anchors (NPCAs); (D) a plurality of collaborationamplifiers (COMs) each hybridized to the SPM and the NPM, wherein eachCOM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and a third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesplice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, aplurality of LMs, and a plurality of LPs, and wherein the SGC provides asignal that is detectable and distinguishable from the background noise.

In another embodiment, the invention provides a kit for in situdetection of a spliced target nucleic acid in a sample of fixed andpermeabilized cells, comprising (A) at least one reagent forpermeabilizing cells; (B) a set of target hybridizing probes comprisinga splice site probe (SP) comprising a target anchor segment (SPAT)capable of hybridizing to a region of the target nucleic acid comprisingthe splice site, and, a neighbor probe (NP) comprising a target anchorsegment (NPAT) capable of hybridizing to a region of the target nucleicacid adjacent to the binding site of the SP; (C) a set of pre-amplifierscomprising an SP pre-amplifier (SPM) comprising a segment capable ofhybridizing to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) comprisinga segment capable of hybridizing to the NP, wherein the NPM comprises aplurality of NP collaboration anchors (NPCAs); (D) a collaborationamplifier (COM) capable of hybridizing to the SPM and the NPM, whereinthe COM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and a third segment comprising aplurality of label amplifier anchor segments; (E) a label amplifier (LM)capable of hybridizing to the label amplifier anchor segment of the COM,wherein the LM comprises a plurality of label probe anchor segments; and(F) a label probe (LP) capable of hybridizing to the label probe anchorsegment of the LM, wherein the LP comprises a detectable label; wherein,upon contacting a sample of fixed and permeabilized cells comprising acell containing a target nucleic acid with the splice site, thecomponents in aforesaid (B)-(F) form a signal generating complex (SGC)comprising the target nucleic acid with the splice site, an SP, an NP,an SPM, an NPM, a plurality of COMs, a plurality of LMs, and a pluralityof LPs, and wherein the SGC provides a signal that is detectable anddistinguishable from the background noise.

Previously described in situ nucleic acid detection methods havedifficulty detecting nucleic acid sequences shorter than 300 to 150bases. In the present invention, since a detectable signal can begenerated with a single SGC and the combined length of SPAT and NPAT canbe as short as 20 bases, the methods of the invention can be readilyadapted to detect short or substantially broken nucleic acid sequence,such as microRNA, micro-insertion or micro-deletion. For detection ofshort or substantially broken nucleic acid sequence, target probe (TP)sets, analogous to the SP-NP sets described herein, are designed tohybridize specifically to adjacent, non-overlapping regions on thetarget nucleic acid, similar to the function of target probe setscontaining both SP and NP, as described herein. In general, the segmentof the target probe that binds to the target nucleic acid, TPAT,analogous to SPAT and NPAT, have the same or similar length. If thetarget sequence is longer, multiple TP sets can be used with multipleSGCs as shown in FIG. 11. The use of multiple SGCs in this situation canbe used either to increase the detection sensitivity or to increase thedetection robustness since any one of the multiple SGCs successfullyformed on the target can generate a detectable signal. Suchconfigurations are particularly useful in detecting long but highlydegraded nucleic acid targets. Many sets of TPs can be designed to becomplementary to non-overlapping regions of the target nucleic acid.Because the target nucleic acid is in a highly degraded state, only alimited number of short segments are accessible. A detectable signal canbe generated as long as one TP pair can be successfully hybridized tothe target sequence. FIG. 11 depicts three exemplary configurations. InFIG. 11A, there is one TP pair in an SGC. The maximum number of SGCsthat can be accommodated has to be at least multiple lengths ofSPAT+NPAT. In FIG. 11B, at least one TP of one SGC contains two TPATsegments that bind to non-overlapping regions of the target nucleicacid, analogous to the integration of an SP and NP into a single probe.In the configuration depicted in FIG. 11B, the maximum number of SGCswithin the same length of target sequence can be increased compared tothe configuration shown in FIG. 11A, improving the sensitivity and/orrobustness of the detection. All single SGC configuration and componentorientation options shown in FIGS. 1 to 8 can similarly be expanded to amultiple SGC setting to detect longer target sequences. For example,FIG. 11C depicts multiple SGCs in a similar configuration to that shownin FIG. 8A when the spatial flexibility of nucleic acid molecules aretaken into consideration.

In many applications, it is highly desirable to detect multiple targetsin the same assay. This present invention provides two differentapproaches to meet this requirement. The first approach is pooling,where, as shown in FIG. 12A, a SGC is assembled for each target, inwhich unique NPAT and SPAT are provided for each target, and NPAP andSPAP are the same across different NP and SP sets for each target. Thisallows all other components of the SGC, that is, NPM, SPM, COM and LPS,to be the same for all SGCs. The pooling provides one detectable signalwhen any one of multiple targets are present in the sample and is veryuseful when a group of targets provide the same or similar biological orclinical utility. The second approach is multiplexing, where, as shownin FIG. 12B, a SGC is assembled for each target and components of eachSGC are unique for that SGC and sequences of these components aredesigned to ensure that there is no cross-hybridization betweendifferent SGCs. Since the label probe (LP) in each SGC has a unique,distinguishable label, a different, uniquely distinguishable signal canbe detected for the presence of each target. The multiplexing approachis useful in applications where different targets provide differentbiological or clinical utility. It is understood that the pooling andmultiplexing approaches can be used in combination to detect multiplegroups of targets.

In another embodiment, the invention provides a method of in situdetection of a target nucleic acid, wherein the target nucleic acid is300 or fewer bases in length, in a sample of fixed and permeabilizedcells. Such a method can comprise the steps of (A) contacting the samplewith a set of target probes (TPs), wherein the set comprises at leasttwo target probes, wherein each TP comprises a target anchor segment(TPAT) that can specifically hybridize to a region of the target nucleicacid and a pre-amplifier anchor segment (TPAP), wherein the setcomprises pairs of TPs that can bind to adjacent, non-overlappingregions of the target nucleic acid; (B) contacting the sample with a setof TP pre-amplifiers (TPMs), wherein the set comprises at least one pairof TPMs, wherein each TPM comprises a segment that can bind to onemember of the pair of TPs that bind to adjacent regions of the targetnucleic acid, and wherein each TPM comprises two or more TPcollaboration anchors (TPCAs); (C) contacting the sample with acollaboration amplifier (COM), wherein the COM comprises a first segmentcomplementary to the TPCA of one member of the pair of TPMs, a secondsegment complementary to the TPCA of the second member of the pair ofTPMs, and a third segment comprising a plurality of label amplifieranchor segments; (D) contacting the sample with a label probe system(LPS), wherein the LPS comprises a plurality of label amplifiers (LMs)and a plurality of label probes (LPs), wherein each LM comprises asegment that can bind to a label amplifier anchor segment of the COM anda plurality of label probe anchor segments, wherein each LP comprises adetectable label and a segment that hybridizes to the label probe anchorsegment of LM, wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid, at leastone pair of TPs, at least one pair of TPMs, a plurality of COMs, aplurality of LMs, and a plurality of LPs; and (E) detecting in situsignal from the SGC on the sample.

In a particular embodiment, the target probe set comprises a splice siteprobe, SP and a neighbor probe, NP. The SP comprises a SPAT capable ofhybridizing to a region of the target nucleic acid containing a splicesite, J, and another region, SPAP, capable of hybridizing to a segmenton the SP pre-amplifier (SPM). The NP comprises an NPAT capable ofhybridizing to a region of the target nucleic acid adjacent to theregion hybridized to SPAT and another region, NPAP, capable ofhybridizing to a segment on the NP pre-amplifier (NPM). The SPM and NPMadditionally comprise one or more segments of SPCAs and NPCAs,respectively. One or more collaboration amplifiers, COM, are provided,wherein each comprises a first segment complementary to the SPCA, asecond segment complementary to the NPCA, and a third segment comprisinga plurality of label amplifier anchor segments. Each COM can attach toNPM and SPM stably when, and only when, the NPCA and SPCA are presentclose together so that it hybridizes to NPCA and SPCA collaboratively.Multiple label amplifiers (LM) capable of hybridizing to the labelamplifier anchor segment of the COM are provided, wherein the LMcomprises a plurality of label probe anchor segments. Multiple labelprobes (LP) are provided capable of hybridizing to the label probeanchor segment of the LM, wherein the LP comprises a detectable label.The SPM, NPM, COM, LM and LP form a signal generating complex (SGC) thatcontain sufficient number of labels capable being detected. Each SGC canappear in an imaging system as a signal focus. If the specific splicejunction does not exist, that is, the two target segments are notspliced together, SP will not hybridize adjacent to NP, SPM will not bepresent close to NPM, which will not allow COM to be stably captured tothe target, and SGC will not be formed; thus, no signal can be detected.

In one embodiment, the invention provides method of in situ detection ofa nucleotide variation of a target nucleic acid in a sample of fixed andpermeabilized cells, comprising: (A) contacting the sample with anucleotide variation probe (SP) and a neighbor probe (NP), wherein theSP comprises a target anchor segment (SPAT) that can specificallyhybridize to a region of the target nucleic acid comprising thenucleotide variation and a pre-amplifier anchor segment (SPAP), andwherein the NP comprises a target anchor segment (NPAT) that canhybridize to a region of the target nucleic acid adjacent to the bindingsite of the SP and a pre-amplifier anchor segment (NPAP); (B) contactingthe sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM),wherein the SPM comprises a segment that can bind to the SP andcomprises two or more SP collaboration anchors (SPCAs), and wherein theNPM comprises a segment that can bind to the NP and comprises two ormore NP collaboration anchors (NPCAs); (C) contacting the sample with acollaboration amplifier (COM), wherein the COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments; (D) contacting the sample with a label probe system (LPS),wherein the LPS comprises a plurality of label amplifiers (LMs) and aplurality of label probes (LPs), wherein each LM comprises a segmentthat can bind to a label amplifier anchor segment of the COM and aplurality of label probe anchor segments, wherein each LP comprises adetectable label and a segment that hybridizes to the label probe anchorsegment of LM, wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising a target nucleic acid with thenucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs,a plurality of LMs, and a plurality of LPs; and (E) detecting in situsignal from the SGC on the sample.

In another embodiment, the invention provides a sample of fixed andpermeabilized cells, comprising: (A) at least one fixed andpermeabilized cell containing a target nucleic acid with a nucleotidevariation; (B) a nucleotide variation probe (SP) comprising a targetanchor segment (SPAT) hybridized to a region of the target nucleic acidcomprising the nucleotide variation, and, a neighbor probe (NP)comprising a target anchor segment (NPAT) hybridized to a region of thetarget nucleic acid adjacent to the binding site of the SP; (C) an SPpre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises aplurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier(NPM) hybridized to the NP, wherein the NPM comprises a plurality of NPcollaboration anchors (NPCAs); (D) a plurality of collaborationamplifiers (COMs) each hybridized to the SPM and the NPM, wherein eachCOM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thenucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs,a plurality of LMs, and a plurality of LPs, and wherein the SGC providesa signal that is detectable and distinguishable from the backgroundnoise.

In another embodiment, the invention provides a tissue slide,comprising: (A) a slide having immobilized thereon a plurality of fixedand permeabilized cells comprising at least one fixed and permeabilizedcell containing a target nucleic acid with a nucleotide variation; (B) anucleotide variation probe (SP) comprising a target anchor segment(SPAT) hybridized to a region of the target nucleic acid comprising thenucleotide variation, and, a neighbor probe (NP) comprising a targetanchor segment (NPAT) hybridized to a region of the target nucleic acidadjacent to the binding site of the SP; (C) an SP pre-amplifier (SPM)hybridized to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridizedto the NP, wherein the NPM comprises a plurality of NP collaborationanchors (NPCAs); (D) a plurality of collaboration amplifiers (COMs) eachhybridized to the SPM and the NPM, wherein each COM comprises a firstsegment complementary to the SPCA, a second segment complementary to theNPCA, and a third segment comprising a plurality of label amplifieranchor segments; (E) a plurality of label amplifiers (LMs) eachhybridized to a label amplifier anchor segment of the COM, wherein theLM comprises a plurality of label probe anchor segments; and (F) aplurality of label probes (LPs) each hybridized to a label probe anchorsegment of the LM, wherein the LP comprises a detectable label; whereinthe aforesaid hybridizations form a signal generating complex (SGC)comprising the target nucleic acid with the nucleotide variation, an SP,an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and aplurality of LPs, and wherein the SGC provides a signal that isdetectable and distinguishable from the background noise.

In another embodiment, the invention provides a kit for in situdetection of a nucleotide variation of a target nucleic acid in a sampleof fixed and permeabilized cells, comprising: (A) at least one reagentfor permeabilizing cells; (B) a set of target hybridizing probescomprising a nucleotide variation probe (SP) comprising a target anchorsegment (SPAT) capable of hybridizing to a region of the target nucleicacid comprising the nucleotide variation, and, a neighbor probe (NP)comprising a target anchor segment (NPAT) capable of hybridizing to aregion of the target nucleic acid adjacent to the binding site of theSP; (C) a set of pre-amplifiers comprising an SP pre-amplifier (SPM)comprising a segment capable of hybridizing to the SP, wherein the SPMcomprises a plurality of SP collaboration anchors (SPCAs), and, an NPpre-amplifier (NPM) comprising a segment capable of hybridizing to theNP, wherein the NPM comprises a plurality of NP collaboration anchors(NPCAs); (D) a collaboration amplifier (COM) capable of hybridizing tothe SPM and the NPM, wherein the COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments; (E) a label amplifier (LM) capable of hybridizing to the labelamplifier anchor segment of the COM, wherein the LM comprises aplurality of label probe anchor segments; and (F) a label probe (LP)capable of hybridizing to the label probe anchor segment of the LM,wherein the LP comprises a detectable label; wherein, upon contacting asample of fixed and permeabilized cells comprising a cell containing atarget nucleic acid with the nucleotide variation, the components inaforesaid (B)-(F) form a signal generating complex (SGC) comprising thetarget nucleic acid with the nucleotide variation, an SP, an NP, an SPM,an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs,and wherein the SGC provides a signal that is detectable anddistinguishable from the background noise. In particular embodiments ofthe above described embodiments of the invention, the nucleotidevariation is selected from the group consisting of a single nucleotidevariation, a multi-nucleotide variation, a splice site, an insertion, adeletion, a rearrangement, and the like.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Detection of Point Mutations

This example describes two exemplary applications of the invention indetecting point mutations in tissue samples.

FIG. 13 shows detection of BRAF mRNA in sections of formalin fixed andparaffin embedded (FFPE) pellet of melanoma cell lines. Melanoma celllines negative (CHL-1, a and a′) and positive (SK-MEL-28, b and b′) forthe V600E point mutation of BRAF were assayed. Cells were hybridized toa target probe system (TPS) containing the wild type detection probe(WDP, sequence: gagatttcA*ctgtagc (SEQ ID NO: 1), A* is a BNA modifiedbase) (a and b) and a TPS containing a BRAF V600E mutation detectionprobe (MDP, sequence: gagatttcT*ctgtagc (SEQ ID NO: 2), T* is a BNAmodified base) (a′ and b′) separately. An SGC configurationsubstantially similar to the one shown in FIG. 1 was used. Signal withprobe targeting wild type BRAF was observed in wild type cells only (a)while V600E mutation was detected in V600E positive cells only withV600E MDP (b′).

FIG. 14 shows the effect of SPATs of various lengths, where an SGCconfiguration substantially similar to the one shown in FIG. 1 was used.In FIG. 14A, CHL-1 cells (no V600E mutation) were hybridized with aV600E mutation probe having a 22 nucleotide (nt) SPAT and showed falsepositive signal. Thus, FIG. 14A shows a high number of false positiveswith the use of a long SPAT. In FIG. 14B, SK-MEL-28 cells containing aV600E mutation were hybridized with a 15 nt SPAT V600E mutation probe,and it showed specific signals. Thus, FIG. 14B shows that the assay hashigher specificity with shorter SPAT.

FIG. 15 shows the effect of including modified bases in a SPAT, where anSGC configuration substantially similar to the one shown in FIG. 1 wasused. In FIG. 15A, SK-MEL-28 cells (containing V600E mutation) werehybridized with a 16 nt SPAT V600E probe with normal bases. FIG. 15Athus shows staining results with normal bases in SPAT. In FIG. 15B,SK-MEL-28 cells were hybridized with a 16 nt SPAT V600E mutation probecontaining a single modified BNA base complementary to the mutation andshowed improved sensitivity with more signals (dots). FIG. 15B thusshows improved results with modified bases used in SPAT.

FIG. 16 shows detection of BRAF mRNA in 2 FFPE colon cancer tissuesknown to be negative (a and a′) and positive (b and b′) for the V600Epoint mutation, where an SGC configuration substantially similar to theone shown in FIG. 1 was used. While signals were observed in bothsamples with probe targeting wild type BRAF mRNA (a and b), V600Emutation mRNA was detected only in the mutation positive sample withprobe designed specifically for V600E mutation (b′). The presence of WDPsignals in the second sample indicated that it had a heterozygous BRAFV600E mutation. In all SNV probes designed in the examples shown in FIG.16, a single BNA modified base was incorporated in SPAT at the locationcomplementary to the mutation site.

FIG. 17 shows the detection of very low abundant and degraded RNA. Thetarget was HGF mRNA, which is more than 1000 nt in length but was knownto be expressed at a very low level. In addition, the RNA was known tobe at least partially degraded in this sample, which is lung cancer informalin-fixed, paraffin embedded (FFPE) tissue sections. FIG. 17A showsthe staining image using methods as described in U.S. Pat. Nos.7,709,198 and 8,658,361 and a target probe set containing 30 pairs ofTPs. The detection sensitivity was low due to a combination of lowexpression and partial RNA degradation. FIG. 17B shows improved stainingusing the method of the invention with 30 pairs of TPs and an SGCconfiguration similar to that shown in FIG. 11A.

FIG. 18 demonstrates that the invention disclosed herein providesenhanced performance in detection of short nucleic acid targets comparedto previously disclosed methods. In FIG. 18A, the detection systemdisclosed in U.S. Pat. Nos. 7,709,198 and 8,658,361 was used with asingle pair of target probes to detect an approximately 50 nt sequenceon POLR2A mRNA in Hela cell pellet. FIG. 18A(a) represents the signalgenerated using POLR2A target probe. FIG. 18A(a′) represents thebackground level generated using a target probe against dapB, a negativecontrol gene. In FIG. 18B, a single SGC with a configuration similar tothat shown in FIG. 11A was used to detect the same target in the samesample type. FIG. 18B(b) represents the signal generated using one TPpair targeting the same sequence of POLR2A mRNA. FIG. 18B(b′) representsthe background level generated targeting dapB, a negative control gene.

The target probe pair in the previously disclosed methods and the methodshown in FIG. 18B have the same configuration, that is, each probecomprises a segment binding to the target sequence and another segmentbinding to a member of the amplification system. In previously disclosedmethods, this member is the preamplifier/amplifier (that is, two targetprobes in the pair bind to the same preamplifier/amplifier molecule). Inthe specific embodiment of the invention used in FIG. 18B, one member ofthe probe pair binds to NPM, the other binds to SPM. The target bindingsegments of the probe pair in both methods are the same. The differenceoccurs in the other segment. In the previously disclosed methods, theother segment (binding to the preamplifier/amplifier) is short, so thatthe preamplifier/amplifier binds to a single probe in the pair unstably.When and only when both members of the target probe pair are presentnext to each other, the preamplifier/amplifier would bind to both probescollaboratively in a stable status. In this specific embodiment for themethod of the invention, the other section is longer, providing for NPMor SPM to bind NP and SP stably. No collaborative hybridization occursbetween SP-SPM or NP-NPM. The collaborative hybridization in the methodof the invention in this particular example shown in FIG. 18B occursbetween COM and SPM+NPM.

FIG. 19 demonstrates in situ detection of specific splice junctions,which can be used to identify a specific splice variant. Cell line H596is known to be META14 positive, that is, exon 14 in the MET gene is“skipped”, resulting in exon 15 splicing with exon 13 in MET RNA. Cellline A549 is the wild-type having all exons 12-15 in MET RNA. Probestargeting splicing junctions E12/13, E13/14 and E14/15 were used todetect the presence of corresponding junctions in FFPE (formalin fixedand paraffin embedded) cell pellets of H596 and A549 cells. The stainingimages are shown in FIG. 19, which show sensitive and specific detectionof targeted splice junctions of E13/15 in H596 cells and E14/15 in A549cells, showing that the META14 splice variant was correctly identified.

These results demonstrate that the methods of utilizing collaborativehybridization can be used to detect single nucleotide variations in atarget nucleic acid in an in situ assay.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

What is claimed is:
 1. A method of in situ detection of a singlenucleotide variation of a target nucleic acid in a sample of fixed andpermeabilized cells, comprising: (A) contacting the sample with a singlenucleotide variation probe (SP) and a neighbor probe (NP), wherein theSP comprises a target anchor segment (SPAT) that can specificallyhybridize to a region of the target nucleic acid comprising the singlenucleotide variation and a pre-amplifier anchor segment (SPAP), andwherein the NP comprises a target anchor segment (NPAT) that canhybridize to a region of the target nucleic acid adjacent to the bindingsite of the SP and a pre-amplifier anchor segment (NPAP); (B) contactingthe sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM),wherein the SPM comprises a segment that can bind to the SP andcomprises two or more SP collaboration anchors (SPCAs), and wherein theNPM comprises a segment that can bind to the NP and comprises two ormore NP collaboration anchors (NPCAs); (C) contacting the sample with acollaboration amplifier (COM), wherein the COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments; (D) contacting the sample with a label probe system (LPS),wherein the LPS comprises a plurality of label amplifiers (LMs) and aplurality of label probes (LPs), wherein each LM comprises a segmentthat can bind to a label amplifier anchor segment of the COM and aplurality of label probe anchor segments, wherein each LP comprises adetectable label and a segment that hybridizes to the label probe anchorsegment of LM, wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising a target nucleic acid with thesingle nucleotide variation, an SP, an NP, an SPM, an NPM, a pluralityof COMs, a plurality of LMs, and a plurality of LPs; and (E) detectingin situ signal from the SGC on the sample.
 2. The method of claim 1,wherein the target nucleic acid is RNA.
 3. The method of claim 1 or 2,wherein the fixed and permeabilized cells are on a tissue slide.
 4. Themethod of any one of claims 1-3, wherein the SPAT is 10 to 20nucleotides in length.
 5. The method of any one of claims 1-4, whereinthe SPAP is 14 to 28 nucleotides in length.
 6. The method of any one ofclaims 1-5, wherein the SP optionally comprises a spacer between theSPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides in length.7. The method of any one of claims 1-6, wherein the NPAT is 16 to 30nucleotides in length.
 8. The method of any one of claims 1-7, whereinthe NPAP is 14 to 28 nucleotides in length.
 9. The method of any one ofclaims 1-8, wherein the NP optionally comprises a spacer between theNPAT and the NPAP, wherein the spacer is 1 to 10 nucleotides in length.10. The method of any one of claims 1-9, wherein the SPM is 50 to 500nucleotides in length.
 11. The method of any one of claims 1-10, whereinthe NPM is 50 to 500 nucleotides in length.
 12. The method of any one ofclaims 1-11, wherein the SPCA is 10 to 20 nucleotides in length.
 13. Themethod of any one of claims 1-12, wherein the SPM optionally comprises aspacer between the two or more SPCA, wherein the spacer between the SPCAis independently 1 to 10 nucleotides in length.
 14. The method of anyone of claims 1-13, wherein the NPCA is 10 to 20 nucleotides in length.15. The method of any one of claims 1-14, wherein the NPM optionallycomprises a spacer between the two or more NPCA, wherein the spacerbetween the NPCA is independently 1 to 10 nucleotides in length.
 16. Themethod of any one of claims 1-15, wherein the COM is 60 to 900nucleotides in length.
 17. The method of any one of claims 1-16, whereinthe COM optionally comprises a spacer between the first, second and/orthird segments, wherein the spacer between the first, second and/orthird segments is independently 1 to 10 nucleotides in length.
 18. Themethod of any one of claims 1-17, wherein the third segment of the COMoptionally comprises a spacer between the plurality of label amplifieranchor segments, wherein the spacer is independently 1 to 10 nucleotidesin length.
 19. The method of any one of claims 1-18, wherein theplurality of COMs bound to the SPM and NPM is in the range of 2 to 20.20. The method of any one of claims 1-19, wherein the plurality of LMsbound to the COM is in the range of 2 to
 20. 21. The method of any oneof claims 1-20, wherein the plurality of LPs bound to the LM is in therange of 2 to
 20. 22. A sample of fixed and permeabilized cells,comprising: (A) at least one fixed and permeabilized cell containing atarget nucleic acid with a single nucleotide variation; (B) a singlenucleotide variation probe (SP) comprising a target anchor segment(SPAT) hybridized to a region of the target nucleic acid comprising thesingle nucleotide variation, and, a neighbor probe (NP) comprising atarget anchor segment (NPAT) hybridized to a region of the targetnucleic acid adjacent to the binding site of the SP; (C) an SPpre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises aplurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier(NPM) hybridized to the NP, wherein the NPM comprises a plurality of NPcollaboration anchors (NPCAs); (D) a plurality of collaborationamplifiers (COMs) each hybridized to the SPM and the NPM, wherein eachCOM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesingle nucleotide variation, an SP, an NP, an SPM, an NPM, a pluralityof COMs, a plurality of LMs, and a plurality of LPs, and wherein the SGCprovides a signal that is detectable and distinguishable from thebackground noise.
 23. A tissue slide, comprising: (A) a slide havingimmobilized thereon a plurality of fixed and permeabilized cellscomprising at least one fixed and permeabilized cell containing a targetnucleic acid with a single nucleotide variation; (B) a single nucleotidevariation probe (SP) comprising a target anchor segment (SPAT)hybridized to a region of the target nucleic acid comprising the singlenucleotide variation, and, a neighbor probe (NP) comprising a targetanchor segment (NPAT) hybridized to a region of the target nucleic acidadjacent to the binding site of the SP; (C) an SP pre-amplifier (SPM)hybridized to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridizedto the NP, wherein the NPM comprises a plurality of NP collaborationanchors (NPCAs); (D) a plurality of collaboration amplifiers (COMs) eachhybridized to the SPM and the NPM, wherein each COM comprises a firstsegment complementary to the SPCA, a second segment complementary to theNPCA, and a third segment comprising a plurality of label amplifieranchor segments; (E) a plurality of label amplifiers (LMs) eachhybridized to a label amplifier anchor segment of the COM, wherein theLM comprises a plurality of label probe anchor segments; and (F) aplurality of label probes (LPs) each hybridized to a label probe anchorsegment of the LM, wherein the LP comprises a detectable label; whereinthe aforesaid hybridizations form a signal generating complex (SGC)comprising the target nucleic acid with the single nucleotide variation,an SP, an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs,and a plurality of LPs, and wherein the SGC provides a signal that isdetectable and distinguishable from the background noise.
 24. A kit forin situ detection of a single nucleotide variation of a target nucleicacid in a sample of fixed and permeabilized cells, comprising: (A) atleast one reagent for permeabilizing cells; (B) a set of targethybridizing probes comprising a single nucleotide variation probe (SP)comprising a target anchor segment (SPAT) capable of hybridizing to aregion of the target nucleic acid comprising the single nucleotidevariation, and, a neighbor probe (NP) comprising a target anchor segment(NPAT) capable of hybridizing to a region of the target nucleic acidadjacent to the binding site of the SP; (C) a set of pre-amplifierscomprising an SP pre-amplifier (SPM) comprising a segment capable ofhybridizing to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) comprisinga segment capable of hybridizing to the NP, wherein the NPM comprises aplurality of NP collaboration anchors (NPCAs); (D) a collaborationamplifier (COM) capable of hybridizing to the SPM and the NPM, whereinthe COM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and a third segment comprising aplurality of label amplifier anchor segments; (E) a label amplifier (LM)capable of hybridizing to the label amplifier anchor segment of the COM,wherein the LM comprises a plurality of label probe anchor segments; and(F) a label probe (LP) capable of hybridizing to the label probe anchorsegment of the LM, wherein the LP comprises a detectable label; wherein,upon contacting a sample of fixed and permeabilized cells comprising acell containing a target nucleic acid with the single nucleotidevariation, the components in aforesaid (B)-(F) form a signal generatingcomplex (SGC) comprising the target nucleic acid with the singlenucleotide variation, an SP, an NP, an SPM, an NPM, a plurality of COMs,a plurality of LMs, and a plurality of LPs, and wherein the SGC providesa signal that is detectable and distinguishable from the backgroundnoise.
 25. The sample of claim 22, the slide of claim 23, or the kit ofclaim 24, wherein the target nucleic acid is RNA.
 26. The sample, slideor kit of any one of claims 22-25, wherein the fixed and permeabilizedcells are on a tissue slide.
 27. The sample, slide or kit of any one ofclaims 22-26, wherein the SPAT is 10 to 20 nucleotides in length. 28.The sample, slide or kit of any one of claims 22-27, wherein the SPAP is14 to 28 nucleotides in length.
 29. The sample, slide or kit of any oneof claims 22-28, wherein the SP optionally comprises a spacer betweenthe SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotides inlength.
 30. The sample, slide or kit of any one of claims 22-29, whereinthe NPAT is 16 to 30 nucleotides in length.
 31. The sample, slide or kitof any one of claims 22-30, wherein the NPAP is 14 to 28 nucleotides inlength.
 32. The sample, slide or kit of any one of claims 22-31, whereinthe NP optionally comprises a spacer between the NPAT and the NPAP,wherein the spacer is 1 to 10 nucleotides in length.
 33. The sample,slide or kit of any one of claims 22-32, wherein the SPM is 50 to 500nucleotides in length.
 34. The sample, slide or kit of any one of claims22-33, wherein the NPM is 50 to 500 nucleotides in length.
 35. Thesample, slide or kit of any one of claims 22-34, wherein the SPCA is 10to 20 nucleotides in length.
 36. The sample, slide or kit of any one ofclaims 22-35, wherein the SPM optionally comprises a spacer between thetwo or more SPCA, wherein the spacer between the SPCA is independently 1to 10 nucleotides in length.
 37. The sample, slide or kit of any one ofclaims 22-36, wherein the NPCA is 10 to 20 nucleotides in length. 38.The sample, slide or kit of any one of claims 22-37, wherein the NPMoptionally comprises a spacer between the two or more NPCA, wherein thespacer between the NPCA is independently 1 to 10 nucleotides in length.39. The sample, slide or kit of any one of claims 22-38, wherein the COMis 60 to 900 nucleotides in length.
 40. The sample, slide or kit of anyone of claims 22-39, wherein the COM optionally comprises a spacerbetween the first, second and/or third segments, wherein the spacerbetween the first, second and/or third segments is independently 1 to 10nucleotides in length.
 41. The sample, slide or kit of any one of claims22-40, wherein the third segment of the COM optionally comprises aspacer between the plurality of label amplifier anchor segments, whereinthe spacer is independently 1 to 10 nucleotides in length.
 42. Thesample, slide or kit of any one of claims 22-41, wherein the pluralityof COMs bound to the SPM and NPM is in the range of 2 to
 20. 43. Thesample, slide or kit of any one of claims 22-42, wherein the pluralityof LMs bound to the COM is in the range of 2 to
 20. 44. The sample,slide or kit of any one of claims 22-43, wherein the plurality of LPsbound to the LM is in the range of 2 to
 20. 45. A method of in situdetection of a target nucleic acid, wherein the target nucleic acid is300 or fewer bases in length, in a sample of fixed and permeabilizedcells, comprising: (A) contacting the sample with a set of target probes(TPs), wherein the set comprises at least two target probes, whereineach TP comprises a target anchor segment (TPAT) that can specificallyhybridize to a region of the target nucleic acid and a pre-amplifieranchor segment (TPAP), wherein the set comprises pairs of TPs that canbind to adjacent, non-overlapping regions of the target nucleic acid;(B) contacting the sample with a set of TP pre-amplifiers (TPMs),wherein the set comprises at least one pair of TPMs, wherein each TPMcomprises a segment that can bind to one member of the pair of TPs thatbind to adjacent regions of the target nucleic acid, and wherein eachTPM comprises two or more TP collaboration anchors (TPCAs); (C)contacting the sample with a collaboration amplifier (COM), wherein theCOM comprises a first segment complementary to the TPCA of one member ofthe pair of TPMs, a second segment complementary to the TPCA of thesecond member of the pair of TPMs, and a third segment comprising aplurality of label amplifier anchor segments; (D) contacting the samplewith a label probe system (LPS), wherein the LPS comprises a pluralityof label amplifiers (LMs) and a plurality of label probes (LPs), whereineach LM comprises a segment that can bind to a label amplifier anchorsegment of the COM and a plurality of label probe anchor segments,wherein each LP comprises a detectable label and a segment thathybridizes to the label probe anchor segment of LM, wherein theaforesaid hybridizations form a signal generating complex (SGC)comprising the target nucleic acid, at least one pair of TPs, at leastone pair of TPMs, a plurality of COMs, a plurality of LMs, and aplurality of LPs; and (E) detecting in situ signal from the SGC on thesample.
 46. A method of in situ detection of a spliced target nucleicacid in a sample of fixed and permeabilized cells, comprising: (A)contacting the sample with a splice site probe (SP) and a neighbor probe(NP), wherein the SP comprises a target anchor segment (SPAT) that canspecifically hybridize to a region of the target nucleic acid comprisingthe splice site and a pre-amplifier anchor segment (SPAP), and whereinthe NP comprises a target anchor segment (NPAT) that can hybridize to aregion of the target nucleic acid adjacent to the binding site of the SPand a pre-amplifier anchor segment (NPAP); (B) contacting the samplewith an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM), whereinthe SPM comprises a segment that can bind to the SP and comprises two ormore SP collaboration anchors (SPCAs), and wherein the NPM comprises asegment that can bind to the NP and comprises two or more NPcollaboration anchors (NPCAs); (C) contacting the sample with acollaboration amplifier (COM), wherein the COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments; (D) contacting the sample with a label probe system (LPS),wherein the LPS comprises a plurality of label amplifiers (LMs) and aplurality of label probes (LPs), wherein each LM comprises a segmentthat can bind to a label amplifier anchor segment of the COM and aplurality of label probe anchor segments, wherein each LP comprises adetectable label and a segment that hybridizes to the label probe anchorsegment of LM, wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising a target nucleic acid with thesplice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, aplurality of LMs, and a plurality of LPs; and (E) detecting in situsignal from the SGC on the sample.
 47. The method of claim 46, whereinthe SPAT can specifically hybridize to one of the two spliced nucleicacid segments.
 48. The method of claim 46, wherein the SPAT canspecifically hybridize to both of the two spliced nucleic acid segments.49. The method of any one of claims 46-48, wherein the target nucleicacid is RNA.
 50. The method of any one of claims 46-49, wherein thefixed and permeabilized cells are on a tissue slide.
 51. The method ofany one of claims 46-50, wherein the SPAT is 10 to 20 nucleotides inlength.
 52. The method of any one of claims 46-51, wherein the SPAP is14 to 28 nucleotides in length.
 53. The method of any one of claims46-52, wherein the SP optionally comprises a spacer between the SPAT andthe SPAP, wherein the spacer is 1 to 10 nucleotides in length.
 54. Themethod of any one of claims 46-53, wherein the NPAT is 16 to 30nucleotides in length.
 55. The method of any one of claims 46-54,wherein the NPAP is 14 to 28 nucleotides in length.
 56. The method ofany one of claims 46-55, wherein the NP optionally comprises a spacerbetween the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotidesin length.
 57. The method of any one of claims 46-56, wherein the SPM is50 to 500 nucleotides in length.
 58. The method of any one of claims46-57, wherein the NPM is 50 to 500 nucleotides in length.
 59. Themethod of any one of claims 46-58, wherein the SPCA is 10 to 20nucleotides in length.
 60. The method of any one of claims 46-59,wherein the SPM optionally comprises a spacer between the two or moreSPCA, wherein the spacer between the SPCA is independently 1 to 10nucleotides in length.
 61. The method of any one of claims 46-60,wherein the NPCA is 10 to 20 nucleotides in length.
 62. The method ofany one of claims 46-61, wherein the NPM optionally comprises a spacerbetween the two or more NPCA, wherein the spacer between the NPCA isindependently 1 to 10 nucleotides in length.
 63. The method of any oneof claims 46-62, wherein the COM is 60 to 900 nucleotides in length. 64.The method of any one of claims 46-63, wherein the COM optionallycomprises a spacer between the first, second and/or third segments,wherein the spacer between the first, second and/or third segments isindependently 1 to 10 nucleotides in length.
 65. The method of any oneof claims 46-64, wherein the third segment of the COM optionallycomprises a spacer between the plurality of label amplifier anchorsegments, wherein the spacer is independently 1 to 10 nucleotides inlength.
 66. The method of any one of claims 46-65, wherein the pluralityof COMs bound to the SPM and NPM is in the range of 2 to
 20. 67. Themethod of any one of claims 46-66, wherein the plurality of LMs bound tothe COM is in the range of 2 to
 20. 68. The method of any one of claims46-67, wherein the plurality of LPs bound to the LM is in the range of 2to
 20. 69. A sample of fixed and permeabilized cells, comprising: (A) atleast one fixed and permeabilized cell containing a spliced targetnucleic acid; (B) a splice site probe (SP) comprising a target anchorsegment (SPAT) hybridized to a region of the target nucleic acidcomprising the splice site, and, a neighbor probe (NP) comprising atarget anchor segment (NPAT) hybridized to a region of the targetnucleic acid adjacent to the binding site of the SP; (C) an SPpre-amplifier (SPM) hybridized to the SP, wherein the SPM comprises aplurality of SP collaboration anchors (SPCAs), and, an NP pre-amplifier(NPM) hybridized to the NP, wherein the NPM comprises a plurality of NPcollaboration anchors (NPCAs); (D) a plurality of collaborationamplifiers (COMs) each hybridized to the SPM and the NPM, wherein eachCOM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and third segment comprising aplurality of label amplifier anchor segments; (E) a plurality of labelamplifiers (LMs) each hybridized to a label amplifier anchor segment ofthe COM, wherein the LM comprises a plurality of label probe anchorsegments; and (F) a plurality of label probes (LPs) each hybridized to alabel probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein the aforesaid hybridizations form a signalgenerating complex (SGC) comprising the target nucleic acid with thesplice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, aplurality of LMs, and a plurality of LPs, and wherein the SGC provides asignal that is detectable and distinguishable from the background noise.70. A tissue slide, comprising: (A) a slide having immobilized thereon aplurality of fixed and permeabilized cells comprising at least one fixedand permeabilized cell containing a spliced target nucleic acid; (B) asplice site probe (SP) comprising a target anchor segment (SPAT)hybridized to a region of the target nucleic acid comprising the splicesite, and, a neighbor probe (NP) comprising a target anchor segment(NPAT) hybridized to a region of the target nucleic acid adjacent to thebinding site of the SP; (C) an SP pre-amplifier (SPM) hybridized to theSP, wherein the SPM comprises a plurality of SP collaboration anchors(SPCAs), and, an NP pre-amplifier (NPM) hybridized to the NP, whereinthe NPM comprises a plurality of NP collaboration anchors (NPCAs); (D) aplurality of collaboration amplifiers (COMs) each hybridized to the SPMand the NPM, wherein each COM comprises a first segment complementary tothe SPCA, a second segment complementary to the NPCA, and a thirdsegment comprising a plurality of label amplifier anchor segments; (E) aplurality of label amplifiers (LMs) each hybridized to a label amplifieranchor segment of the COM, wherein the LM comprises a plurality of labelprobe anchor segments; and (F) a plurality of label probes (LPs) eachhybridized to a label probe anchor segment of the LM, wherein the LPcomprises a detectable label; wherein the aforesaid hybridizations forma signal generating complex (SGC) comprising the target nucleic acidwith the splice site, an SP, an NP, an SPM, an NPM, a plurality of COMs,a plurality of LMs, and a plurality of LPs, and wherein the SGC providesa signal that is detectable and distinguishable from the backgroundnoise.
 71. A kit for in situ detection of a spliced target nucleic acidin a sample of fixed and permeabilized cells, comprising: (A) at leastone reagent for permeabilizing cells; (B) a set of target hybridizingprobes comprising a splice site probe (SP) comprising a target anchorsegment (SPAT) capable of hybridizing to a region of the target nucleicacid comprising the splice site, and, a neighbor probe (NP) comprising atarget anchor segment (NPAT) capable of hybridizing to a region of thetarget nucleic acid adjacent to the binding site of the SP; (C) a set ofpre-amplifiers comprising an SP pre-amplifier (SPM) comprising a segmentcapable of hybridizing to the SP, wherein the SPM comprises a pluralityof SP collaboration anchors (SPCAs), and, an NP pre-amplifier (NPM)comprising a segment capable of hybridizing to the NP, wherein the NPMcomprises a plurality of NP collaboration anchors (NPCAs); (D) acollaboration amplifier (COM) capable of hybridizing to the SPM and theNPM, wherein the COM comprises a first segment complementary to theSPCA, a second segment complementary to the NPCA, and a third segmentcomprising a plurality of label amplifier anchor segments; (E) a labelamplifier (LM) capable of hybridizing to the label amplifier anchorsegment of the COM, wherein the LM comprises a plurality of label probeanchor segments; and (F) a label probe (LP) capable of hybridizing tothe label probe anchor segment of the LM, wherein the LP comprises adetectable label; wherein, upon contacting a sample of fixed andpermeabilized cells comprising a cell containing a target nucleic acidwith the splice site, the components in aforesaid (B)-(F) form a signalgenerating complex (SGC) comprising the target nucleic acid with thesplice site, an SP, an NP, an SPM, an NPM, a plurality of COMs, aplurality of LMs, and a plurality of LPs, and wherein the SGC provides asignal that is detectable and distinguishable from the background noise.72. The sample of claim 69, the slide of claim 70, or the kit of claim71, wherein the SPAT can specifically hybridize to one of the twospliced nucleic acid segments.
 73. The sample, slide or kit of any oneof claims 69-72, wherein the SPAT can specifically hybridize to both ofthe two spliced nucleic acid segments.
 74. The sample, slide or kit ofany one of claims 69-73, wherein the target nucleic acid is RNA.
 75. Thesample, slide or kit of any one of claims 69-74, wherein the fixed andpermeabilized cells are on a tissue slide.
 76. The sample, slide or kitof any one of claims 69-75, wherein the SPAT is 10 to 20 nucleotides inlength.
 77. The sample, slide or kit of any one of claims 69-76, whereinthe SPAP is 14 to 28 nucleotides in length.
 78. The sample, slide or kitof any one of claims 69-77, wherein the SP optionally comprises a spacerbetween the SPAT and the SPAP, wherein the spacer is 1 to 10 nucleotidesin length.
 79. The sample, slide or kit of any one of claims 69-78,wherein the NPAT is 16 to 30 nucleotides in length.
 80. The sample,slide or kit of any one of claims 69-79, wherein the NPAP is 14 to 28nucleotides in length.
 81. The sample, slide or kit of any one of claims69-80, wherein the NP optionally comprises a spacer between the NPAT andthe NPAP, wherein the spacer is 1 to 10 nucleotides in length.
 82. Thesample, slide or kit of any one of claims 69-81, wherein the SPM is 50to 500 nucleotides in length.
 83. The sample, slide or kit of any one ofclaims 69-82, wherein the NPM is 50 to 500 nucleotides in length. 84.The sample, slide or kit of any one of claims 69-83, wherein the SPCA is10 to 20 nucleotides in length.
 85. The sample, slide or kit of any oneof claims 69-84, wherein the SPM optionally comprises a spacer betweenthe two or more SPCA, wherein the spacer between the SPCA isindependently 1 to 10 nucleotides in length.
 86. The sample, slide orkit of any one of claims 69-85, wherein the NPCA is 10 to 20 nucleotidesin length.
 87. The sample, slide or kit of any one of claims 69-86,wherein the NPM optionally comprises a spacer between the two or moreNPCA, wherein the spacer between the NPCA is independently 1 to 10nucleotides in length.
 88. The sample, slide or kit of any one of claims69-87, wherein the COM is 60 to 900 nucleotides in length.
 89. Thesample, slide or kit of any one of claims 69-88, wherein the COMoptionally comprises a spacer between the first, second and/or thirdsegments, wherein the spacer between the first, second and/or thirdsegments is independently 1 to 10 nucleotides in length.
 90. The sample,slide or kit of any one of claims 69-89, wherein the third segment ofthe COM optionally comprises a spacer between the plurality of labelamplifier anchor segments, wherein the spacer is independently 1 to 10nucleotides in length.
 91. The sample, slide or kit of any one of claims69-90, wherein the plurality of COMs bound to the SPM and NPM is in therange of 2 to
 20. 92. The sample, slide or kit of any one of claims69-91, wherein the plurality of LMs bound to the COM is in the range of2 to
 20. 93. The sample, slide or kit of any one of claims 69-92,wherein the plurality of LPs bound to the LM is in the range of 2 to 20.94. A method of in situ detection of a nucleotide variation of a targetnucleic acid in a sample of fixed and permeabilized cells, comprising:(A) contacting the sample with a nucleotide variation probe (SP) and aneighbor probe (NP), wherein the SP comprises a target anchor segment(SPAT) that can specifically hybridize to a region of the target nucleicacid comprising the nucleotide variation and a pre-amplifier anchorsegment (SPAP), and wherein the NP comprises a target anchor segment(NPAT) that can hybridize to a region of the target nucleic acidadjacent to the binding site of the SP and a pre-amplifier anchorsegment (NPAP); (B) contacting the sample with an SP pre-amplifier (SPM)and an NP pre-amplifier (NPM), wherein the SPM comprises a segment thatcan bind to the SP and comprises two or more SP collaboration anchors(SPCAs), and wherein the NPM comprises a segment that can bind to the NPand comprises two or more NP collaboration anchors (NPCAs); (C)contacting the sample with a collaboration amplifier (COM), wherein theCOM comprises a first segment complementary to the SPCA, a secondsegment complementary to the NPCA, and a third segment comprising aplurality of label amplifier anchor segments; (D) contacting the samplewith a label probe system (LPS), wherein the LPS comprises a pluralityof label amplifiers (LMs) and a plurality of label probes (LPs), whereineach LM comprises a segment that can bind to a label amplifier anchorsegment of the COM and a plurality of label probe anchor segments,wherein each LP comprises a detectable label and a segment thathybridizes to the label probe anchor segment of LM, wherein theaforesaid hybridizations form a signal generating complex (SGC)comprising a target nucleic acid with the nucleotide variation, an SP,an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and aplurality of LPs; and (E) detecting in situ signal from the SGC on thesample.
 95. The method of claim 94, wherein the nucleotide variation isselected from the group consisting of a single nucleotide variation, amulti-nucleotide variation, a splice site, an insertion, a deletion, anda rearrangement.
 96. The method of claim 94 or 95, wherein the targetnucleic acid is RNA.
 97. The method of any one of claims 94-96, whereinthe fixed and permeabilized cells are on a tissue slide.
 98. The methodof any one of claims 1-97, wherein the SPAT is 10 to 20 nucleotides inlength.
 99. The method of any one of claims 1-98, wherein the SPAP is 14to 28 nucleotides in length.
 100. The method of any one of claims 1-99,wherein the SP optionally comprises a spacer between the SPAT and theSPAP, wherein the spacer is 1 to 10 nucleotides in length.
 101. Themethod of any one of claims 1-100, wherein the NPAT is 16 to 30nucleotides in length.
 102. The method of any one of claims 1-101,wherein the NPAP is 14 to 28 nucleotides in length.
 103. The method ofany one of claims 1-102, wherein the NP optionally comprises a spacerbetween the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotidesin length.
 104. The method of any one of claims 1-103, wherein the SPMis 50 to 500 nucleotides in length.
 105. The method of any one of claims1-104, wherein the NPM is 50 to 500 nucleotides in length.
 106. Themethod of any one of claims 1-105, wherein the SPCA is 10 to 20nucleotides in length.
 107. The method of any one of claims 1-106,wherein the SPM optionally comprises a spacer between the two or moreSPCA, wherein the spacer between the SPCA is independently 1 to 10nucleotides in length.
 108. The method of any one of claims 1-107,wherein the NPCA is 10 to 20 nucleotides in length.
 109. The method ofany one of claims 1-108, wherein the NPM optionally comprises a spacerbetween the two or more NPCA, wherein the spacer between the NPCA isindependently 1 to 10 nucleotides in length.
 110. The method of any oneof claims 1-109, wherein the COM is 60 to 900 nucleotides in length.111. The method of any one of claims 1-110, wherein the COM optionallycomprises a spacer between the first, second and/or third segments,wherein the spacer between the first, second and/or third segments isindependently 1 to 10 nucleotides in length.
 112. The method of any oneof claims 1-111, wherein the third segment of the COM optionallycomprises a spacer between the plurality of label amplifier anchorsegments, wherein the spacer is independently 1 to 10 nucleotides inlength.
 113. The method of any one of claims 1-112, wherein theplurality of COMs bound to the SPM and NPM is in the range of 2 to 20.114. The method of any one of claims 1-113, wherein the plurality of LMsbound to the COM is in the range of 2 to
 20. 115. The method of any oneof claims 1-114, wherein the plurality of LPs bound to the LM is in therange of 2 to
 20. 116. A sample of fixed and permeabilized cells,comprising: (A) at least one fixed and permeabilized cell containing atarget nucleic acid with a nucleotide variation; (B) a nucleotidevariation probe (SP) comprising a target anchor segment (SPAT)hybridized to a region of the target nucleic acid comprising thenucleotide variation, and, a neighbor probe (NP) comprising a targetanchor segment (NPAT) hybridized to a region of the target nucleic acidadjacent to the binding site of the SP; (C) an SP pre-amplifier (SPM)hybridized to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridizedto the NP, wherein the NPM comprises a plurality of NP collaborationanchors (NPCAs); (D) a plurality of collaboration amplifiers (COMs) eachhybridized to the SPM and the NPM, wherein each COM comprises a firstsegment complementary to the SPCA, a second segment complementary to theNPCA, and third segment comprising a plurality of label amplifier anchorsegments; (E) a plurality of label amplifiers (LMs) each hybridized to alabel amplifier anchor segment of the COM, wherein the LM comprises aplurality of label probe anchor segments; and (F) a plurality of labelprobes (LPs) each hybridized to a label probe anchor segment of the LM,wherein the LP comprises a detectable label; wherein the aforesaidhybridizations form a signal generating complex (SGC) comprising thetarget nucleic acid with the nucleotide variation, an SP, an NP, an SPM,an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs,and wherein the SGC provides a signal that is detectable anddistinguishable from the background noise.
 117. A tissue slide,comprising: (A) a slide having immobilized thereon a plurality of fixedand permeabilized cells comprising at least one fixed and permeabilizedcell containing a target nucleic acid with a nucleotide variation; (B) anucleotide variation probe (SP) comprising a target anchor segment(SPAT) hybridized to a region of the target nucleic acid comprising thenucleotide variation, and, a neighbor probe (NP) comprising a targetanchor segment (NPAT) hybridized to a region of the target nucleic acidadjacent to the binding site of the SP; (C) an SP pre-amplifier (SPM)hybridized to the SP, wherein the SPM comprises a plurality of SPcollaboration anchors (SPCAs), and, an NP pre-amplifier (NPM) hybridizedto the NP, wherein the NPM comprises a plurality of NP collaborationanchors (NPCAs); (D) a plurality of collaboration amplifiers (COMs) eachhybridized to the SPM and the NPM, wherein each COM comprises a firstsegment complementary to the SPCA, a second segment complementary to theNPCA, and a third segment comprising a plurality of label amplifieranchor segments; (E) a plurality of label amplifiers (LMs) eachhybridized to a label amplifier anchor segment of the COM, wherein theLM comprises a plurality of label probe anchor segments; and (F) aplurality of label probes (LPs) each hybridized to a label probe anchorsegment of the LM, wherein the LP comprises a detectable label; whereinthe aforesaid hybridizations form a signal generating complex (SGC)comprising the target nucleic acid with the nucleotide variation, an SP,an NP, an SPM, an NPM, a plurality of COMs, a plurality of LMs, and aplurality of LPs, and wherein the SGC provides a signal that isdetectable and distinguishable from the background noise.
 118. A kit forin situ detection of a nucleotide variation of a target nucleic acid ina sample of fixed and permeabilized cells, comprising: (A) at least onereagent for permeabilizing cells; (B) a set of target hybridizing probescomprising a nucleotide variation probe (SP) comprising a target anchorsegment (SPAT) capable of hybridizing to a region of the target nucleicacid comprising the nucleotide variation, and, a neighbor probe (NP)comprising a target anchor segment (NPAT) capable of hybridizing to aregion of the target nucleic acid adjacent to the binding site of theSP; (C) a set of pre-amplifiers comprising an SP pre-amplifier (SPM)comprising a segment capable of hybridizing to the SP, wherein the SPMcomprises a plurality of SP collaboration anchors (SPCAs), and, an NPpre-amplifier (NPM) comprising a segment capable of hybridizing to theNP, wherein the NPM comprises a plurality of NP collaboration anchors(NPCAs); (D) a collaboration amplifier (COM) capable of hybridizing tothe SPM and the NPM, wherein the COM comprises a first segmentcomplementary to the SPCA, a second segment complementary to the NPCA,and a third segment comprising a plurality of label amplifier anchorsegments; (E) a label amplifier (LM) capable of hybridizing to the labelamplifier anchor segment of the COM, wherein the LM comprises aplurality of label probe anchor segments; and (F) a label probe (LP)capable of hybridizing to the label probe anchor segment of the LM,wherein the LP comprises a detectable label; wherein, upon contacting asample of fixed and permeabilized cells comprising a cell containing atarget nucleic acid with the nucleotide variation, the components inaforesaid (B)-(F) form a signal generating complex (SGC) comprising thetarget nucleic acid with the nucleotide variation, an SP, an NP, an SPM,an NPM, a plurality of COMs, a plurality of LMs, and a plurality of LPs,and wherein the SGC provides a signal that is detectable anddistinguishable from the background noise.
 119. The sample of claim 116,the slide of claim 117, or the kit of claim 118, wherein the nucleotidevariation is selected from the group consisting of a single nucleotidevariation, a multi-nucleotide variation, a splice site, an insertion, adeletion, and a rearrangement.
 120. The sample, slide or kit of any oneof claims 116-119, wherein the target nucleic acid is RNA.
 121. Thesample, slide or kit of any one of claims 116-120, wherein the fixed andpermeabilized cells are on a tissue slide.
 122. The sample, slide or kitof any one of claims 116-121, wherein the SPAT is 10 to 20 nucleotidesin length.
 123. The sample, slide or kit of any one of claims 116-122,wherein the SPAP is 14 to 28 nucleotides in length.
 124. The sample,slide or kit of any one of claims 116-123, wherein the SP optionallycomprises a spacer between the SPAT and the SPAP, wherein the spacer is1 to 10 nucleotides in length.
 125. The sample, slide or kit of any oneof claims 116-124, wherein the NPAT is 16 to 30 nucleotides in length.126. The sample, slide or kit of any one of claims 116-125, wherein theNPAP is 14 to 28 nucleotides in length.
 127. The sample, slide or kit ofany one of claims 116-126, wherein the NP optionally comprises a spacerbetween the NPAT and the NPAP, wherein the spacer is 1 to 10 nucleotidesin length.
 128. The sample, slide or kit of any one of claims 116-127,wherein the SPM is 50 to 500 nucleotides in length.
 129. The sample,slide or kit of any one of claims 116-128, wherein the NPM is 50 to 500nucleotides in length.
 130. The sample, slide or kit of any one ofclaims 116-129, wherein the SPCA is 10 to 20 nucleotides in length. 131.The sample, slide or kit of any one of claims 116-130, wherein the SPMoptionally comprises a spacer between the two or more SPCA, wherein thespacer between the SPCA is independently 1 to 10 nucleotides in length.132. The sample, slide or kit of any one of claims 116-131, wherein theNPCA is 10 to 20 nucleotides in length.
 133. The sample, slide or kit ofany one of claims 116-132, wherein the NPM optionally comprises a spacerbetween the two or more NPCA, wherein the spacer between the NPCA isindependently 1 to 10 nucleotides in length.
 134. The sample, slide orkit of any one of claims 116-133, wherein the COM is 60 to 900nucleotides in length.
 135. The sample, slide or kit of any one ofclaims 116-134, wherein the COM optionally comprises a spacer betweenthe first, second and/or third segments, wherein the spacer between thefirst, second and/or third segments is independently 1 to 10 nucleotidesin length.
 136. The sample, slide or kit of any one of claims 116-135,wherein the third segment of the COM optionally comprises a spacerbetween the plurality of label amplifier anchor segments, wherein thespacer is independently 1 to 10 nucleotides in length.
 137. The sample,slide or kit of any one of claims 116-136, wherein the plurality of COMsbound to the SPM and NPM is in the range of 2 to
 20. 138. The sample,slide or kit of any one of claims 116-137, wherein the plurality of LMsbound to the COM is in the range of 2 to
 20. 139. The sample, slide orkit of any one of claims 116-138, wherein the plurality of LPs bound tothe LM is in the range of 2 to 20.